Method for generation of immunoglobulin sequences

ABSTRACT

The present invention relates to a method for generating immunoglobulin sequences against cell-associated antigens, more particularly, antigens that are membrane-anchored. The invention also provides immunoglobulin sequences obtainable by the method of the invention. Specifically, the present invention relates to the generation of immunoglobulin sequences by use of DNA vaccination. More specifically, the present invention relates to generation of immunoglobulin sequences in camelids, preferably directed against cell-associated antigens, in particular antigens with multiple transmembrane spanning domains, including GPCRs and ion channels, by DNA vaccination. Furthermore, the present invention relates to said immunoglobulin sequences against cell-associated antigens, more particularly, antigens that are membrane-anchored, such as e.g. GPCRs and ion channels, more preferably ion channels.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 12/643,286, filed on Dec.21, 2009, which claims the benefit under 35 U.S.C. 119(e) of U.S.provisional application Ser. No. 61/203,188, filed Dec. 19, 2008, theentire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for generating immunoglobulinsequences against cell-associated antigens, more particularly, antigensthat are membrane-anchored. The invention also provides immunoglobulinsequences obtainable by the method of the invention. Specifically, thepresent invention relates to the generation of immunoglobulin sequencesby use of DNA vaccination. More specifically, the present inventionrelates to generation of immunoglobulin sequences in camelids,preferably directed against cell-associated antigens, in particularantigens with multiple transmembrane spanning domains, including GPCRsand ion channels, by DNA vaccination. Furthermore, the present inventionrelates to said immunoglobulin sequences against cell-associatedantigens, more particularly, antigens that are membrane-anchored, suchas e.g. GPCRs and ion channels, more preferably ion channels.

BACKGROUND OF THE INVENTION

Cell-associated antigens, more specifically those with single ormultiple transmembrane domains, are difficult to purify in their nativeconformation. In order to identify antibodies (or antibody fragmentssuch as Nanobodies) against native epitopes which are able to modify thefunction of the target in vivo, it is crucial to administer the targetantigen in its native conformation to the camelid [Dormitz et al.(2008). Trends in Biotechnology 26: 659-667]. In absence of purifiednative protein of these cell-associated antigens, the most appliedimmunization strategy consists of repetitive injections of whole cellsfunctionally expressing the antigen of choice at regular intervals.Examples of targets for which such immunization strategy has beenexecuted successfully (i.e. resulting in the identification ofneutralizing, in vivo matured Nanobodies) are described in WO 05/044858and WO 07/042289). Repeated booster injections of target expressingcells, however, often result in diluted or non-detectable targetspecific immune responses, especially when the expression level of thetarget is low and the host cell background is highly immunogenic. Thehumoral response can be focused more towards the target by using a cellline of camelid origin, which is less immunogenic to llama.Nevertheless, repeated injections of (quasi)self-surface markers alsoresult in a response against the camelid cell line surface markers.

The identification of (neutralizing) selective antibodies against GPCRs,Ion channels or any other type of multispanning cell surface marker ischallenging [Michel et al. (2009). Naunyn-Schmied Archives Pharmacology379:385-388], since i) most often no native protein is available forimmunization or subsequent antibody identification, ii) multispannersoften show low immunogenicity (due to a limited number of extracellularsurface exposed amino acid residues compared to most singletransmembrane receptors) and iii) multispanning surface molecules areoften expressed at low densities.

SUMMARY OF THE INVENTION

Immunoglobulin sequences, such as antibodies and antigen bindingfragments derived therefrom are widely used to specifically target theirrespective antigens in research and therapeutic applications. Typically,the generation of antibodies involves the immunization of experimentalanimals, fusion of antibody producing cells to create hybridomas andscreening for the desired specificities. Alternatively, antibodies canbe generated by screening of naïve or synthetic libraries e.g. by phagedisplay.

The generation of immunoglobulin sequences, such as Nanobodies, has beendescribed extensively in various publications, among which WO 94/04678,Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 can beexemplified. In these methods, camelids are immunized with the targetantigen in order to induce an immune response against said targetantigen. The repertoire of Nanobodies obtained from said immunization isfurther screened for Nanobodies that bind the target antigen.

In these instances, the generation of antibodies requires purifiedantigen for immunization and/or screening. Antigens can be purified fromnatural sources, or in the course of recombinant production.

An important class of potential therapeutic targets are cell associatedantigens, including transmembrane antigens, in particular transmembraneantigens with multiple membrane spanning domains. Cell-associated, andespecially membrane bound antigens, however, are difficult to obtain intheir natural conformation because they are embedded within, or anchoredin the cell membrane. In order to obtain immunoglobulin sequencesagainst epitopes present in the natural conformation, i.e.conformational epitopes, which are present in vivo, it is howeveressential to immunize with the target antigen in the correctconformation. Such conformational epitopes are of paramount importancefor creating pharmaceutically active immunoglobulin sequences. Forexample, an immunoglobulin sequence specifically interacting with thepore region of an ion channel will affect its conductivity, and thusprovide a pharmacological effect.

Immunization and/or screening for immunoglobulin sequences can beperformed using peptide fragments of such antigens. However, such anapproach will not provide antibodies to conformation dependent epitopes,as such epitopes cannot be reproduced by short synthetic peptides.

Therefore, for these cell-associated antigens, immunization with wholecells carrying the antigen and subsequent screening of the repertoire ofNanobodies induced in this way for Nanobodies that bind thecell-associated antigen is an option (as was done e.g. in WO2005/044858; WO 2007/042289; WO 2008/074839; WO 2009/068625; WO2009/138519). However, such cells express a multitude of antigens,resulting in an antibody response that is largely directed to antigensof no interest. Hence, the antibody response obtainable by this approachis characterized by a low specificity, and in particular by a very lowfrequency of the antibodies of interest amongst all antibodiesgenerated. Hence this approach precludes the efficient generation ofantibodies to the target antigen of interest.

Hence, the art provides no satisfactory method to generate specificantibody responses of suitable breadth against conformational epitopes,in particular of membrane associated antigens.

It is the objective of the present invention to overcome theseshortcomings of the art. In particular it is an objective of the presentinvention to provide a method for creating immunoglobulin sequencesagainst complex antigens, like cell associated antigens that exhibitconformational epitopes.

The above mentioned problems are overcome by the present invention. Ithas been found that genetic vaccination can result in an antibodyresponse of good specificity and acceptable breadth againstconformational epitopes, i.e. against cell associated antigens in theirnatural conformation.

The present invention relates to the following.

A method for the generation of immunoglobulin sequences that can bind toand/or have affinity for a cell-associated antigen comprising the stepsof:

a) genetic vaccination of a non-human animal with a nucleic acidencoding said cell-associated antigen or a domain or specific part ofsaid cell associated antigen; and

b) optionally boosting the animal with said antigen in its naturalconformation selected from cells comprising natural or transfected cellsexpressing the cell-associated antigen, cell derived membrane extracts,vesicles or any other membrane derivative harbouring enriched antigen,liposomes, or virus particles expressing the cell associated antigenc) screening a set, collection or library of immunoglobulin sequencesderived from said non-human animal for immunoglobulin sequences that canbind to and/or have affinity for said cell-associated antigen. In aparticular embodiment of the invention, said cell-associated antigen isselected from transmembrane antigens, transmembrane antigens withmultiple spanning domains, such as GPCRs or ion channels. According tothe invention said non-human animal can be selected from vertebrate,shark, mammal, lizard, camelid, llama, preferably camelids and llama.

In one embodiment of the invention, the immunoglobulin sequences arelight chain variable domain sequences (e.g. a V_(L)-sequence), or heavychain variable domain sequences (e.g. a V_(H)-sequence); morespecifically, the immunoglobulin sequences can be heavy chain variabledomain sequences that are derived from a conventional four-chainantibody or heavy chain variable domain sequences that are derived froma heavy chain antibody.

According to the invention, the immunoglobulin sequences can be domainantibodies, or immunoglobulin sequences that are suitable for use asdomain antibodies, single domain antibodies, or immunoglobulin sequencesthat are suitable for use as single domain antibodies, “dAbs”, orimmunoglobulin sequences that are suitable for use as dAbs, orNanobodies, including but not limited to V_(HH) sequences, andpreferably are Nanobodies.

According to the invention, vaccination can be performed by aneedle-free jet injection, by a ballistic method, by needle-mediatedinjections such as Tattoo, by topical application of the DNA onto theskin in patches or by any of these administration methods followed by invivo electroporation, and furthermore includes vaccination performed byintradermal, intramuscular or subcutaneous administration of DNA.

The set, collection or library of immunoglobulin sequences can beobtained from the blood of said non-human mammal.

In the present invention, said cell-associated antigen can be expressedon any cell background which allows expression of the nativeconformation of the antigen. Examples of such cell backgrounds are Cho,Cos7, Hek293, or cells of camelid origin. Preferably, saidcell-associated antigen is a membrane-spanning antigen, including butnot limited to an antigen selected from CXCR7, CXCR4 and P2X7.

The set, collection or library of immunoglobulin sequences can beexpressed on a set, collection or sample of cells or viruses and saidset, collection or sample of cells or viruses is screened for cells orviruses that express an immunoglobulin sequence that can bind to and/orhave affinity for said cell-associated antigen, more specifically, anucleic acid sequence that encodes the immunoglobulin sequence that canbind to and/or has affinity for said cell-associated antigen can bepurified and/or isolated from the cell or virus, followed by expressionof said immunoglobulin sequence.

According to the invention, the set, collection or library ofimmunoglobulin sequences can be encoded by a set, collection or libraryof nucleic acid sequences and said set, collection or library of nucleicacid sequences is screened for nucleic acid sequences that encode animmunoglobulin sequence that can bind to and/or have affinity for saidcell-associated antigen; more specifically, the nucleic acid sequencesthat encode an immunoglobulin sequence that can bind to and/or haveaffinity for said cell-associated antigen can be purified and/orisolated, followed by expressing said immunoglobulin sequence.

According to the invention, the immunoglobulin sequence that can bind toand/or has affinity for said cell-associated antigen can be purifiedand/or isolated.

The invention also relates to immunoglobulin obtainable by a method asdescribed herein, and compositions comprising the said immunoglobulinsequences, more in particular to immunoglobulin sequence that aredirected against (as defined herein) ion channels and GPCRs.

In particular, the present invention relates to immunoglobulin sequencesthat are directed against (as defined herein) ion channels, as well asto compounds or constructs, and in particular proteins and polypeptides,that comprise or essentially consist of one or more such immunoglobulinsequences (also referred to herein as “immunoglobulin sequences of theinvention”, “compounds of the invention”, and “polypeptides of theinvention”, respectively). The invention also relates to nucleic acidsencoding such immunoglobulin sequences and polypeptides (also referredto herein as “nucleic acids of the invention” or “nucleotide sequencesof the invention”); to methods for preparing such immunoglobulinsequences and polypeptides; to host cells expressing or capable ofexpressing such immunoglobulin sequences or polypeptides; tocompositions, and in particular to pharmaceutical compositions, thatcomprise such immunoglobulin sequences, polypeptides, nucleic acidsand/or host cells; and to uses of such immunoglobulin sequences orpolypeptides, nucleic acids, host cells and/or compositions, inparticular for prophylactic, therapeutic or diagnostic purposes, such asthe prophylactic, therapeutic or diagnostic purposes mentioned herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C: Kinetics of humoral immune response in llama followinggenetic vaccination with PigJet (FIGS. 1A and 1B) or Tattoo method(FIGS. 1A and 1C). Sera were tested at 1/400 dilution. Arrows indicatethe moment of Jet immunizations, the Tattoo short-interval regimes areindicated by asterisks.

FIG. 2: Strong secondary serum response observed in DNA vaccinatedllamas after single HBsAg protein boost.

FIG. 3. Humoral immune responses obtained via “DNA” prime—“protein”boost protocol (llamas 124, 160, 117, 203) versus protein immunizations(llamas 32 and 33).

FIG. 4. Heavy chain antibody (IgG2 and 3)-mediated antibody responseagainst HBsAg (llama 124).

FIGS. 5A and 5B. CXCR4 specific staining of HEK293 cells followingpVAX-hCXCR4 transfection and camelid after pcDNA3.1-hCXCR4 transfection.

FIGS. 6A and 6B. CXCR4 specific serum conversion in llamas via geneticimmunization. FIG. 6A: “DNA” prime—“cell” boost protocol; FIG. 6B:“cell” boost protocol only.

FIG. 7. hCXCR4 specific Nanobody discovery efficiency from ‘DNA’, ‘PB’or ‘cell’ repertoires (number of libraries).

FIG. 8. Complementary target specific Nanobody repertoires obtainedafter genetic immunization (DNA) and single cell boost (PB). Numbers ofrepertoire specific Nanobody families are depicted.

FIGS. 9A and 9B. Average binding potencies determined via FACS (FIG. 9A)or ELISA (FIG. 9B) of CXCR4-specific Nanobody repertoire after primaryscreening identified after genetic immunization (‘DNA’), subsequentsingle cell boost (‘PB’) or after complete cell immunization (‘cell’)(number of individual Nanobodies).

FIG. 10. mP2X7-specific serum conversion in llamas via geneticimmunization.

FIG. 11. Sequence alignment of human P2X7 and mouse mP2X7. The humanP2X7 amino acid sequence is SEQ ID NO: 792 and the mouse P2X7 amino acidsequence is SEQ ID NO: 793.

FIG. 12. mP2X7-specific serum conversion in llamas via genetic cocktailimmunization.

FIGS. 13A and 13B. Pre-adsorbed immune sera contain mP2X7-specificheavy-chain antibodies after genetic immunization. FIG. 13A: Anti-LlamaIgG-1; FIG. 13B: Anti-Llama IgG-2/3.

FIG. 14. mP2X7 specific Nanobody discovery efficiency from ‘DNA’, ‘PB’or ‘cell’ repertoires (number of libraries).

FIG. 15. Sequence diversity of Nanobodies identified after primaryscreening for mP2X7. DNA: DNA immunization; PB: DNA immunizationfollowed by post boost.

FIG. 16. Representative example of periplasmatic extracts of clones 2C4and 5A1 with and without preincubation with anti-Myc-antibodies forbinding to mP2X7-expressing Hek293 and control WT Hek293 cells.

FIG. 17. Titration curve of Nanobodies from 24 different families forbinding to mP2X7-Cho cells.

FIG. 18. Dose-dependent inhibition by Nanobodies of ligand-induced CD62Lshedding on Yac-1 cells expressing mP2X7.

FIGS. 19A-19D. Titration of nucleotides in mP2X7-mediatedCD62L-ectodomain shedding with fixed concentration of Nanobodies (2 μM).FIGS. 19A and 19B Nanobodies 14D5, 13G9, 7H6, 13B5, no nb, Art 2.2 nb;FIGS. 19C and 19D: 4B4, 7D6, 13A7, 8G11, 8F5, 8G12, no nb, irrelevantnb, Art2.2 nb.

FIGS. 20A-20D. Bivalent Nanobodies of 13A7 (cell), 8011 (PB) and 14D5(DNA) show enhanced potencies in blocking or enhancing mP2X7 function.FIGS. 20A and 20B: Nanobodies or Nanobody constructs: 8G11,13A7-35GS-13A7, 13A7, 8G11-35GS-8G11, irrelevant nb; FIGS. 20C and 20D:Nanobodies or Nanobody constructs: 14D5, 14D5-35GS-14D5, irrelevant nb.

FIG. 21. Average binding potencies determined via ELISA of mP2X7specific Nanobody repertoire identified after genetic immunization(‘DNA’), subsequent single cell boost (‘PB’) or after complete cellimmunization (‘cell’)(number of unique Nanobodies).

FIG. 22. Sequence alignment of 84 non-redundant mP2X7-selectedNanobodies to human germline sequence VH3-23/JH5 (SEQ ID NOs:705-786).

FIG. 23. CXCR7 specific serum conversion in llamas via Gene Gun geneticimmunization. White or black bars represent the MCF values generated onCXCR7 transfected or non-transfected HEK293 WT cells.

FIG. 24. CXCR7 specific Nanobody discovery efficiency from ‘DNA’, ‘PB’or ‘cell’ repertoires (number of libraries).

FIG. 25. Complementary target specific Nanobody repertoires obtainedafter genetic immunization (DNA) and single cell boost (PB). Numbers ofrepertoire specific Nanobody families are depicted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses, but is not limited to, the subjectmatter of the appended claims.

A) DEFINITIONS

Unless indicated or defined otherwise, all terms used have their usualmeaning in the art, which will be clear to the skilled person. Referenceis for example made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Rot et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10^(th) Ed. Blackwell Publishing, UK (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as to thegeneral background art cited herein;

Unless indicated otherwise, the term “immunoglobulin sequence”—whetherused herein to refer to a heavy chain antibody or to a conventional4-chain antibody—is used as a general term to include both the full-sizeantibody, the individual chains thereof, as well as all parts, domainsor fragments thereof (including but not limited to antigen-bindingdomains or fragments such as V_(HH) domains or V_(H)/V_(L) domains,respectively). The terms antigen-binding molecules or antigen-bindingprotein are used interchangeably with immunoglobulin sequence, andinclude Nanobodies.

In one embodiment of the invention, the immunoglobulin sequences arelight chain variable domain sequences (e.g. a V_(L)-sequence), or heavychain variable domain sequences (e.g. a V_(H)-sequence); morespecifically, the immunoglobulin sequences can be heavy chain variabledomain sequences that are derived from a conventional four-chainantibody or heavy chain variable domain sequences that are derived froma heavy chain antibody.

According to the invention, the immunoglobulin sequences can be domainantibodies, or immunoglobulin sequences that are suitable for use asdomain antibodies, single domain antibodies, or immunoglobulin sequencesthat are suitable for use as single domain antibodies, “dAbs”, orimmunoglobulin sequences that are suitable for use as dAbs, orNanobodies, including but not limited to V_(HH) sequences, andpreferably are Nanobodies.

The immunoglobulin sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more immunoglobulin sequences of theinvention and which may optionally further comprise one or more furtherimmunoglobulin sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or moreimmunoglobulin sequences of the invention may be used as a binding unitin such a protein or polypeptide, which may optionally contain one ormore further immunoglobulin sequences that can serve as a binding unit(i.e. against one or more other targets than cell associated antigens),so as to provide a monovalent, multivalent or multispecific polypeptideof the invention, respectively, all as described herein. Such a proteinor polypeptide may also be in essentially isolated form (as definedherein).

The invention includes immunoglobulin sequences of different origin,comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulinsequences. The invention also includes fully human, humanized orchimeric immunoglobulin sequences. For example, the invention comprisescamelid immunoglobulin sequences and humanized camelid immunoglobulinsequences, or camelized domain antibodies, e.g. camelized Dab asdescribed by Ward et al (see for example WO 94/04678 and Davies andRiechmann (1994 and 1996)). Moreover, the invention comprises fusedimmunoglobulin sequences, e.g. forming a multivalent and/ormultispecific construct (for multivalent and multispecific polypeptidescontaining one or more V_(HH) domains and their preparation, referenceis also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350,2001, as well as to for example WO 96/34103 and WO 99/23221), andimmunoglobulin sequences comprising tags or other functional moieties,e.g. toxins, labels, radiochemicals, etc., which are derivable from theimmunoglobulin sequences of the present invention.

The immunoglobulin sequence and structure of an immunoglobulin sequence,in particular a Nanobody can be considered—without however being limitedthereto—to be comprised of four framework regions or “FR's”, which arereferred to in the art and herein as “Framework region 1” or “FR1”; as“Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and as“Framework region 4” or “FR4”, respectively; which framework regions areinterrupted by three complementary determining regions or “CDR's”, whichare referred to in the art as “Complementarity Determining Region 1” or“CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively.

The total number of amino acid residues in a Nanobody can be in theregion of 110-120, is preferably 112-115, and is most preferably 113. Itshould however be noted that parts, fragments, analogs or derivatives(as further described herein) of a Nanobody are not particularly limitedas to their length and/or size, as long as such parts, fragments,analogs or derivatives meet the further requirements outlined herein andare also preferably suitable for the purposes described herein.

As used herein, the term “immunoglobulin sequences” refers to both thenucleic acid sequences coding for an immunoglobulin molecule, and theimmunoglobulin polypeptide per se. Any more limiting meaning will beapparent from the particular context.

All these molecules are also referred to as “polypeptide of theinvention”, which is synonymous with “immunoglobulin sequences” of theinvention.

In addition, the term “sequence” as used herein (for example in termslike “immunoglobulin sequence”, “antibody sequence”, “variable domainsequence”, “V_(HH) sequence” or “protein sequence”), should generally beunderstood to include both the relevant immunoglobulin sequence as wellas nucleic acid sequences or nucleotide sequences encoding the same,unless the context requires a more limited interpretation.

In the following, reference to a “nucleic acid molecule” of theinvention may either relate to the nucleic acid for genetic vaccination,or the nucleic acid encoding the immunoglobulin sequences of theinvention, or both, as will be apparent from the specific context.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein; as well as to for example thefollowing reviews Presta, Adv. Drug Deliv. Rev, 2006, 58 (5-6): 640-56;Levin and Weiss, Md. Biosyst. 2006, 2(1): 49-57; Irving et al., J.Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000,21 Suppl. A, S106-12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43,which describe techniques for protein engineering, such as affinitymaturation and other techniques for improving the specificity and otherdesired properties of proteins such as immunoglobulins.

The invention relates to immunoglobulin sequences that can bind toand/or have affinity for an antigen as defined herein. In the context ofthe present invention, “binding to and/or having affinity for” a certainantigen has the usual meaning in the art as understood e.g. in thecontext of antibodies and their respective antigens.

In particular embodiments of the invention, the term “binds to and/orhaving affinity for” means that the immunoglobulin sequence specificallyinteracts with an antigen, and is used interchangeably withimmunoglobulin sequences “against” the said antigen.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular immunoglobulinsequence, antigen-binding molecule or antigen-binding protein (such as aNanobody or a polypeptide of the invention) can bind. The specificity ofan antigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (K_(D)), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the K_(D), the stronger the binding strength between anantigenic determinant and the antigen-binding molecule (alternatively,the affinity can also be expressed as the affinity constant (K_(A)),which is 1/K_(D)). As will be clear to the skilled person (for exampleon the basis of the further disclosure herein), affinity can bedetermined in a manner known per se, depending on the specific antigenof interest. Avidity is the measure of the strength of binding betweenan antigen-binding molecule (such as a Nanobody or polypeptide of theinvention) and the pertinent antigen. Avidity is related to both theaffinity between an antigenic determinant and its antigen binding siteon the antigen-binding molecule and the number of pertinent bindingsites present on the antigen-binding molecule. Typically, immunoglobulinsequences of the present invention (such as the immunoglobulinsequences, Nanobodies and/or polypeptides of the invention) will bind totheir antigen with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or lessand more preferably 10⁻⁶ to 10⁻¹² moles/liter (i.e. with an associationconstant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷to 10¹² liter/moles or more and more preferably 10⁸ to 10¹²liter/moles), and/or

bind to cell associated antigens as defined herein with a k_(on)-rate ofbetween 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, suchas between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹; and/or bind to cell associatedantigens as defined herein with a k_(off) rate between 1s⁻¹(t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible complexwith a t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴s⁻¹ and 10⁻⁶ s⁻¹.

Any K_(D) value greater than 10⁻⁴ mol/liter (or any K_(A) value lowerthan 10⁴ M⁻¹) liters/mol is generally considered to indicatenon-specific binding.

Preferably, a monovalent immunoglobulin sequence of the invention willbind to the desired antigen with an affinity less than 500 nM,preferably less than 200 nM, more preferably less than 10 nM, such asless than 500 pM.

Specific binding of an antigen-binding protein to an antigen orantigenic determinant can be determined in any suitable manner known perse, including, for example, Scatchard analysis and/or competitivebinding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA) and sandwich competition assays, and the different variantsthereof known per se in the art; as well as the other techniquesmentioned herein.

The dissociation constant may be the actual or apparent dissociationconstant, as will be clear to the skilled person. Methods fordetermining the dissociation constant will be clear to the skilledperson, and for example include the techniques mentioned herein. In thisrespect, it will also be clear that it may not be possible to measuredissociation constants of more then 10⁻⁴ moles/liter or 10⁻³ moles/liter(e.g., of 10⁻² moles/liter). Optionally, as will also be clear to theskilled person, the (actual or apparent) dissociation constant may becalculated on the basis of the (actual or apparent) association constant(K_(A)), by means of the relationship [K_(D)=1/K_(A)].

The affinity denotes the strength or stability of a molecularinteraction. The affinity is commonly given as by the K_(D), ordissociation constant, which has units of mol/liter (or M). The affinitycan also be expressed as an association constant, K_(A), which equals1/K_(D) and has units of (mol/liter)⁻¹ (or M⁻¹). In the presentspecification, the stability of the interaction between two molecules(such as an immunoglobulin sequence, immunoglobulin sequence, Nanobodyor polypeptide of the invention and its intended target) will mainly beexpressed in terms of the K_(D) value of their interaction; it beingclear to the skilled person that in view of the relation K_(A)=1/K_(D),specifying the strength of molecular interaction by its K_(D) value canalso be used to calculate the corresponding K_(A) value. The K_(D)-valuecharacterizes the strength of a molecular interaction also in athermodynamic sense as it is related to the free energy (DG) of bindingby the well known relation DG=RT·ln(K_(D)) (equivalentlyDG=−RT·ln(K_(A))), where R equals the gas constant, T equals theabsolute temperature and ln denotes the natural logarithm.

The K_(D) for biological interactions, such as the binding of theimmunoglobulin sequences of the invention to the cell associated antigenas defined herein, which are considered meaningful (e.g. specific) aretypically in the range of 10⁻¹⁰M (0.1 nM) to 10⁻⁵M (10000 nM). Thestronger an interaction is, the lower is its K_(D).

The K_(D) can also be expressed as the ratio of the dissociation rateconstant of a complex, denoted as k_(off), to the rate of itsassociation, denoted k_(on) (so that K_(D)=k_(off)/k_(on) andK_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s isthe SI unit notation of second). The on-rate k_(on) has units M⁻¹s⁻¹.

As regards immunoglobulin sequences of the invention, the on-rate mayvary between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, approaching thediffusion-limited association rate constant for bimolecularinteractions. The off-rate is related to the half-life of a givenmolecular interaction by the relation t_(1/2)=ln(2)/k_(off). Theoff-rate of immunoglobulin sequences of the invention may vary between10⁻⁶ s⁻¹ (near irreversible complex with a t_(1/2) of multiple days) to1 s⁻¹ (t_(1/2)=0.69 s).

The affinity of a molecular interaction between two molecules can bemeasured via different techniques known per se, such as the well knownsurface plasmon resonance (SPR) biosensor technique (see for exampleOber et al., Intern. Immunology, 13, 1551-1559, 2001) where one moleculeis immobilized on the biosensor chip and the other molecule is passedover the immobilized molecule under flow conditions yielding k_(on)k_(off) measurements and hence K_(D) (or K_(A)) values. This can forexample be performed using the well-known Biacore instruments.

It will also be clear to the skilled person that the measured K_(D) maycorrespond to the apparent K_(D) if the measuring process somehowinfluences the intrinsic binding affinity of the implied molecules forexample by artefacts related to the coating on the biosensor of onemolecule. Also, an apparent K_(D) may be measured if one moleculecontains more than one recognition sites for the other molecule. In suchsituation the measured affinity may be affected by the avidity of theinteraction by the two molecules.

Another approach that may be used to assess affinity is the 2-step ELISA(Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J.Immunol. Methods, 77, 305-19, 1985). This method establishes a solutionphase binding equilibrium measurement and avoids possible artefactsrelating to adsorption of one of the molecules on a support such asplastic.

However, the accurate measurement of K_(D) may be quite labour-intensiveand as consequence, often apparent K_(D) values are determined to assessthe binding strength of two molecules. It should be noted that as longas all measurements are made in a consistent way (e.g. keeping the assayconditions unchanged) apparent K_(D) measurements can be used as anapproximation of the true K_(D) and hence in the present document K_(D)and apparent K_(D) should be treated with equal importance or relevance.

Finally, it should be noted that in many situations the experiencedscientist may judge it to be convenient to determine the bindingaffinity relative to some reference molecule. For example, to assess thebinding strength between molecules A and B, one may e.g. use a referencemolecule C that is known to bind to B and that is suitably labelled witha fluorophore or chromophore group or other chemical moiety, such asbiotin for easy detection in an ELISA or FACS (Fluorescent activatedcell sorting) or other format (the fluorophore for fluorescencedetection, the chromophore for light absorption detection, the biotinfor streptavidin-mediated ELISA detection). Typically, the referencemolecule C is kept at a fixed concentration and the concentration of Ais varied for a given concentration or amount of B. As a result an IC₅₀value is obtained corresponding to the concentration of A at which thesignal measured for C in absence of A is halved. Provided K_(D ref), theK_(D) of the reference molecule, is known, as well as the totalconcentration c_(ref) of the reference molecule, the apparent K_(D) forthe interaction AB can be obtained from following formula:K_(D)=IC₅₀/(1+c_(rel)/K_(D ref)). Note that if c_(ref)<<K_(D ref),K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in aconsistent way (e.g. keeping c_(ref) fixed) for the binders that arecompared, the strength or stability of a molecular interaction can beassessed by the IC₅₀ and this measurement is judged as equivalent toK_(D) or to apparent K_(D) throughout this text.

In the context of the present invention, “conformation dependentepitope”, or “conformational epitope” denotes an epitope that comprisesamino acids which are not within a single consecutive stretch of theprimary sequence of the antigen. In other words, due to the secondaryand/or tertiary structure of a protein target, amino acids which may bespaced apart in the primary sequence are brought into proximity to eachother and thereby participate in the formation of an epitope. If forexample an antigen comprises three amino acid loops, residues on eachone of these loops may participate in the formation of a single epitope.The same applies to antigens comprising more than one domain or subunit.In this case, an epitope may be formed by amino acids on differentdomains or subunits. Complete or partial denaturing of the protein byappropriate conditions, i.e. the partial or full destruction ofsecondary and/or tertiary structures, will also partly or fully destroyconformational epitopes. The skilled person will understand that theprecise conditions under which a conformational epitope is destroyed bydenaturing a protein will depend on the nature of the protein and thespecific circumstances.

In a preferred embodiment, the present invention is directed toimmunoglobulin sequences against conformational epitopes. In particular,the invention concerns immunoglobulin sequences against conformationalepitopes on cell-associated antigens as defined herein, which maypreferably be camelid immunoglobulin sequences, including Nanobodies.

In the context of the present invention, “cell-associated an en” meansantigens that are firmly anchored in or located within the membranes ofa cell (including membranes of subcellular compartments and organelles),and includes antigens that have a single or multiple transmembraneregions. In other words, the term refers to antigens exhibitingmembrane-dependent conformational epitopes. In particular, the termrefers to antigens having conformational epitopes as defined herein. Theterm encompasses transmembrane antigens, transmembrane antigens withmultiple membrane spanning domains such as GPCRs or ion channels.Amongst all these antigens the skilled person knows a range of druggabletarget antigens, which represent a preferred cell associated antigen ofthe present invention. The invention in particular relates to cellassociated antigens wherein the conformation dependent epitope isdependent on the correct anchoring and/or location in the membrane.Thus, the invention provides immunoglobulin sequences against suchconformation dependent epitopes.

In a preferred embodiment the invention relates to antigens that areintegral membrane proteins having one, or more preferably multiplemembrane spanning domains. These antigens will reside in and operatewithin a cell's plasma membrane, and/or the membranes of subcellularcompartments and organelles. Many transmembrane proteins, such astransmembrane receptors comprise two or more subunits or domains, whichfunctionally interact with one another.

Integral membrane proteins comprise three distinct parts or domains,i.e. an extracellular (or extracompartmental) domain, a transmembranedomain and an intracellular (or intracompartmental) domain. A proteinhaving multiple transmembrane domains will typically also have multipleextra- and intra cellular/compartmental domains. For example, a seventransmembrane receptor comprise seven transmembrane domains.

Thus, the term cell associated antigen as understood herein is intendedto exclude antigens that are only loosely associated, i.e. that are notfirmly anchored or located within a membrane. An antigen is firmlyanchored if it comprises at least one domain or part that extends intothe membrane.

In one embodiment, the invention excludes antigens that have a membraneinsertion via a lipid tail, but no transmembrane domain. In thisinstance, the conformation of the hydrophilic portion or domain of theprotein will not depend on the membrane environment. It will, forexample, be possible to express a recombinant protein lacking the lipidtail, which is in the proper conformation, i.e. expresses theconformational epitopes also present if the antigen is associated withthe membrane via the lipid tail. Similarly, any other proteins which areonly loosely associated are excluded from the invention in a particularembodiment. “Loosely associated” in this connection means proteins whichexhibit their natural conformation even in the absence of membrane, i.e.their natural conformation is not dependent on the anchoring orembedding within a membrane. In a further particular embodiment, theinvention excludes ART2.2.

Typical examples of cell associated antigens according to the inventioncomprise seven membrane domain receptors, including G-protein coupledreceptors, such as the ones further described herein, Adrenergicreceptor, Olfactory receptors, Receptor tyrosine kinases, such asEpidermal growth factor receptor, Insulin Receptor, Fibroblast growthfactor receptors, High affinity neurotrophin receptors, and EphReceptors, Integrins, Low Affinity Nerve Growth Factor Receptor, NMDAreceptor, Several Immune receptors including Toll-like receptor, T cellreceptor and CD28. Furthermore, cell associated antigens according tothe invention comprise also ion channels, such as the ones furtherdescribed herein, calcium channels, sodium channels, potassium channels,2P ion channels, 6-TM ion channels, voltage-gated ion channels and/orcalcium-activated potassium channels.

As used herein, the term “cell-associated antigen” is intended toinclude, and also refer to, any part, fragment, subunit, or domain ofsaid cell associated antigen. Any subsection of the cell associatedantigen falls within the scope of the present invention, provided itrepresents a conformational epitope of interest. If for example theepitope of interest is located in a binding site of a receptor, or thepore of an ion channel, any fragment(s) of the cell associated antigencapable of forming said epitope are included in the invention.Preferably, those parts, domains, fragments or subunits will be thoseparts of the cell associated antigen which are responsible for themembrane-dependent conformation. If for example a protein comprisesseveral transmembrane domains, linked by extended intracellular loops,it is envisaged that such loops are in part or fully omitted, withoutinfluencing the extracellular conformational epitopes.

In particular, the present invention relates to immunoglobulin sequencesdirected to cell associated antigens in their natural conformation. Inthe context of the present invention, “natural conformation” means thatthe protein exhibits its secondary and/or tertiary structure, inparticular its membrane dependent secondary and/or tertiary structure.In other words, the natural conformation describes the protein in anon-denatured form, and describes a conformation wherein theconformational epitopes, in particular the membrane dependentconformational epitopes, are present. Specifically, the protein willhave the conformation that is present when the protein is integratedinto or firmly attached to a membrane. Antigens can be obtained in theirnatural conformation when present in cells comprising natural ortransfected cells expressing the cell-associated antigen, cell derivedmembrane extracts, vesicles or any other membrane derivative harbouringantigen, liposomes, or virus particles expressing the cell associatedantigen. In any of these embodiments, antigen may be enriched bysuitable means. Said cell-associated antigen can be expressed on anysuitable cell allowing expression of the antigen in its native ornatural conformation, encompassing, but not limited to Cho, Cos7,Hek293, or cells of camelid origin.

The cell associated antigen of the present invention is preferably adruggable membrane protein, in particular a druggable membrane proteinhaving multiple membrane spanning domains. In one embodiment of theinvention, the target is a GPCR or an ion channel.

Specific, non limiting examples of ion channels that represent cellassociated antigens according to the present invention are provided inthe following. Also listed are therapeutic effects of immunoglobulinsequences specifically recognizing such ion channels.

1. Two-P potassium channels (see Goldstein et al., PharmacologicalReviews, 57, 4, 527 (2005)), such as K_(2P)1.1, K_(2P)2.1, K_(2P)3.1,K_(2P)3.1, K_(2P)4.1, K_(2P)5.1, K_(2P)6.1, K_(2P)7.1, K_(2P)9.1,K_(2P)10.1, K_(2P)12.1, K_(2P)13.1, K_(2P)15.1, K_(2P)16.1, K_(2P)17.1and K_(2P)18.1, which can all be screened using electrophysiologicalassays such as FLIPR or patch-clamp. 2. CatSper channels (see Claphamand Garbers, Pharmacological Reviews, 57, 4, 451 (2005)), such asCatSper-1 and CatSper-2 (both involved in fertility and sperm motility),CatSper-3 and CatSper-4, which can all be screened usingelectrophysiological assays such as FLIPR, patch-clamp or calciumimaging techniques. 3. Two-pore channels (see Clapham and Garbers,Pharmacological Reviews, 57, 4, 451 (2005)), such as TPC1 and TPC2. 4.Cyclic nucleotide-gated channels (see Hofman et al., PharmacologicalReviews, 57, 4, 455 (2005), such as CNGA-1, CNGA-2, CNGA-3, CNGA-4A,CNGB1 and CNGB3, which can be screened using techniques such as patch-clamp and calcium imaging 5. Hyperpolarization-activated cyclicnucleotide-gated channels (see Hofman et al., Pharmacological Reviews,57, 4, 455 (2005)), such as HCN1, HCN2, HCN3, HCN4 (all regarded aspromising pharmacological targets for development of drugs for cardiacarrhythmias and ischemic heart disease), which can be screened usingtechniques such as voltage-clamp. 6. Inwardly rectifying potassiumchannels (see Kubo et al., Pharmacological Reviews, 57, 4, 509 (2005)),such as K_(ir)1.1, K_(ir)21. K_(ir)2.2, K_(ir)2.3, K_(ir)2.4, K_(ir)3.1,K_(ir)3.2, K_(ir)3.3, K_(ir)3.4, K_(ir)3.4, K_(ir)4.2, K_(ir)5.1,K_(ir)6.1 (a target for antihypertensive agents and coronaryvasodilators), K_(ir)6.2 (the target for pentholamine; its subunit SUR1is a target for the treatment of diabetes and PHHI) and Kir7.1 (which isa possible site for side-effects of calcium channel blockers), which canbe screened using techniques such as voltage-clamp. 7. Calcium-activatedpotassium channels (see Wei et al., Pharmacological Reviews, 57, 4, 463(2005)), such as   K_(Ca)1.1 - openers of which may be useful in thetreatment of stroke,   epilepsy, bladder over-reactivity, asthma,hypertension, gastric   hypermotility and psychoses;   K_(Ca)2.1 -modulators of which may be useful in the treatment of various   diseasessuch as myotonic muscular dystrophy, gastrointestinal   dysmotility,memory disorders, epilepsy, narcolepsy and alcohol   intoxication.Openers of K_(Ca)2.2 have been proposed for cerebellar ataxia;  K_(Ca)2.2 - modulators of which may be useful in the treatment ofvarious   diseases such as myotonic muscular dystrophy, gastrointestinal  dysmotility, memory disorders, epilepsy, narcolepsy and alcohol  intoxication. Openers of K_(Ca)2.2 have been proposed for cerebellarataxia;   K_(Ca)2.2 - modulators of which may be useful in the treatmentof various   diseases such as myotonic muscular dystrophy,gastrointestinal   dysmotility, memory disorders, epilepsy, narcolepsy,hypertension and   urinary incontinence;   K_(Ca)3.1 - blockers of whichmay be useful in the treatment of sickle cell   anemia, diarrhea, asimmunosuppressants, EAE, the prevention of   restenosis andangiogenesis, the treatment of brain injuries and the   reduction ofbrain oedema. Openers if K_(Ca)3.1 have been proposed for the  treatment of cystic fibrosis and COPD; as well as K_(Ca)4.1, K_(Ca)4.2and K_(Ca)5.1; all of which can be screened using electrophysiologicaltechniques or techniques such as patch-clamp or voltage- clamp. 8.Potassium channels (see Shieh et al., Pharmacological Reviews, 57, 4,557 (2005) and Gutman et al., Pharmacological Reviews, 57, 4, 473(2005)), including:   voltage-gated calcium channels such as Kv1.1,Kv1.2, Kv1.3, Kv1.4,   Kv1.5, Kv1.6 and Kv.17;   voltage- and cGMP-gatedcalcium channels such as Kv1.10;   beta-subunits of Kv channels such asKvBeta-1, KvBeta-2 and KvBeta-   3;   Shab-like channels such as Kv2.1and Kv2.2;   Shaw-like channels such as Kv3.1, Kv3.2. Kv3.3 and Kv3.4;  Shal-like channels such as Kv4.1, Kv4.2, Kv4.3, Kv5.1, Kv6.1, Kv6.2,  Kv8.1, Kv9.1, Kv9.2, Kv9.3, KH1 and KH2;   Ether-a-go-go-channels suchas EAG, HERG, BEC1 and BEC2;   MinK-type channels such as MinK, MiRP1and MiRP2;   KvLQT-type channels such as KvLQT1, KvLQT2, KvLQT3, KvLQT4,  KvLQT5   Inwardly rectifying potassium channels such as thosementioned above;   Sulfonylurea receptors such as the sulfonylureareceptors 1 and 2;   Large conductance calcium-activated channels suchas Slo and the   Beta-subunits of BK_(Ca);   Small conductancecalcium-activated channels such as SK1, SK2 and   SK3;   Intermediateconductance calcium-activated channels such as IKCa1;   Two-porepotassium channels such as TWIK1, TREK, TASK, TASK2,   TWIK2, TOSS,TRAAK and CTBAK1; all of which can be screened usingelectrophysiological techniques or techniques such as patch-clamp orvoltage-clamp. Potassium channels are implicated in a wide variety ofdiseases and disorders such as cardiac diseases (such as arrhythmia),neuronal diseases, neuromuscular disorders, hearing and vestibulardiseases, renal diseases, Alzheimer's disease. and metabolic diseases;and are targets for active compounds in these diseases. Reference isagain made to the reviews by Shieh et al. and by Gutman et al. (and thefurther prior art cited therein) as well as to the further referencescited in the present specification. Tables 3 and 4 of the Shieh reviewalso mention a number of known openers and blockers, respectively, ofvarious potassium channels and the disease indications for which theyhave been used/proposed. 9. Voltage-gated calcium channels (seeCatterall et al., Pharmacological Reviews, 57, 4, 411 (2005)), such as:Ca_(v)1.2 - modulators of which are useful as Ca²⁺ antagonists;Ca_(v)1.3 - modulators of which have been proposed for modulating theheart rate, as antidepressants and as drugs for hearing disorders;Ca_(v)2.1 - modulators of which have been proposed as analgesics forinflammatory pain; Ca_(v)2.2 - modulators of which have been proposed asanalgesics for pain such as inflammatory pain, postsurgical pain,thermal hyperalgesia, chronic pain and mechanical allodynia; Ca_(v)3.2 -which has been proposed as a target for epilepsy, hypertension andangina pectoris; Ca_(v)3.3 - which has been proposed as a target for thetreatment of thalamic oscillations; and Ca_(v)1.1, Ca_(v)1.4, Ca_(v)2.3,Ca_(v)3.1,; all of which can be screened using techniques such aspatch-clamp, voltage-clamp and calcium imaging. 10. Transient receptorpotential (TRP) channels (see Clapham et al., Pharmacological Reviews,57, 4, 427 (2005)) such as: TRPC channels such as TRPC1, TRPC2, TRPC3,TRPC4, TRPC5, TRPC6 and TRPC7; TRPV channels such as TRPV1, TRPV2,TRPV3, TRPV4, TRPV5 and TRPV6; TRPM channels such as TRPM1, TRPM2,TRPM3, TRPM4, TRPM5, TRPM6, TRPM7 and TRPM8; TRPA1; TRPP channels suchas PKD1,, PKD2L1 and PKD2L2, which are involved in polycystic kidneydisease; TRPML channels such as mucolipin 1, mucolipin 2 and mucolipin3; which can be screened using techniques such as patch-clamp andcalcium imaging. 11. Voltage-gated sodium channels (see Catterall etal., Pharmacological Reviews, 57, 4, 397 (2005)), such as:   Na_(v)1.1,Na_(v)1.2 and Na_(v)1.3 - which are a target for drugs for the  prevention and treatment of epilepsy and seizures;   Na_(v)1.4 - whichis a target for local anaesthetics for the treatment of   myotonia;  Na_(v)1.5 - which is a target for antiarrhythmic drugs;   Na_(v)1.6 -which is a target for antiepileptic and analgesic drugs;   Na_(v)1.7,Na_(v)1.8 and Na_(v)1.9 - which are potential targets for local  anaesthetics; all of which can be screened using voltage clamp ortechniques involving voltage-sensitive dyes.

Ion channels and the diseases and disorders which they are associatedare well known in the art. Reference is for example made to thefollowing reviews: Goldstein et al., Pharmacological Reviews, 57, 4, 527(2005); Yu et al., Pharmacological Reviews, 57, 4, 387 (2005); Claphamand Garbers, Pharmacological Reviews, 57, 4, 451 (2005); Hoffmann etal., Pharmacological Reviews, 57, 4, 455 (2005); Kubo et al.,Pharmacological Reviews, 57, 4, 509 (2005); Wei et al., PharmacologicalReviews, 57, 4, 463 (2005); Shieh et al, Pharmacological Reviews, 57, 4,557 (2005); Catterall et al, Pharmacological Reviews, 57, 4, 411 (2005);Gutman et al., Pharmacological Reviews, 57, 4, 473 (2005); Clapham etal., Pharmacological Reviews, 57, 4, 427 (2005); and Catterall et al.,Pharmacological Reviews, 57, 4, 397 (2005); as well as the furtherreferences cited in these reviews and the following articles andreviews: Chandy et al., Trends in Pharmacological Sciences, May 2004,280-289; Takana and Shigenobu, J. Pharmacol. Sci., 99, 214-200 (2005);Padinjat and Andrews, Journal of Cell Science, 117, 5707-5709 (2004);Amir et al., The Journal of Pain, Vol. 7, No. 5, S1-S29 (2006); Devor,The Journal of Pain, Vol. 7, No. 15, S3-S12 (2006); Xie et al., CurrentDrug Discovery, April 2004, 31-33; Vianna-Jorge and Suarez-Kurtz,BioDrugs 2004, 18(5), 329-41; Garcia and Kaczorowski, Sci STKE, 2005,302; Gopalakrishnan and Shieh, Expert Opin. Ther. Targets, 2005, 8(5),437-58; Mannhold, Med. Res. Rev., 2004, 24(2), 213-66; Sabido-David etal., Expert Opin. Investig. Drugs, 2004, 13(1) 1249-61; and Christ,Journal of Andrology, Vol. 23, No. 5, S10-S19 (2002), and to the furtherprior art cited therein (all incorporated herein by reference), as wellas to the table below and the further prior art cited in thisapplication.

As can be seen from these reviews and this prior art, ion channels aregenerally classified on the basis of the ions that can flow through them(i.e. as calcium channels, sodium channels or potassium channels), onthe basis of their composition and structure (e.g. the number and typeof subunits, pores and transmembrane domains, for example 2P ionchannels, 6-TM ion channels, etc.), and/or on the basis of the manner inwhich they are activated (e.g. voltage-gated ion channels orcalcium-activated potassium channels).

Ion channels generally comprise a number of transmembrane domainsubunits, linked by a combination of intracellular and extracellularloops. Reference is again made to the references and prior art citedabove, as well as to Benham, Nature Biotechnology, October 2005,1234-1235 and the further prior art cited herein.

The fact that ion channels are membrane proteins, as well as their knownassociation with various disease states, make ion channels attractivemolecular targets for pharmaceutical and veterinary compounds (i.e. forprophylaxis, therapy or diagnosis). Also, methods for screeningpotential pharmaceutical or veterinary compounds for activity (either asagonists, antagonists, blockers and/or openers) and/or selectivity withrespect to ion channels and their biological or physiological activityare well known in the art. Some non-limiting examples of suitabletechniques, depending upon the ion channel involved, include techniquessuch as patch clamp, voltage clamp, measuring ion flux, FLIPR, calciumimaging and electrophysiological techniques.

Specific, non limiting examples of GPCRs that represent cell associatedantigens according to the present invention are provided in thefollowing. Also listed are some exemplary therapeutic effects ofimmunoglobulin sequences of the present invention that are directedagainst these GPCRs.

Class A GPCRs

-   -   Acetylcholine receptor (agonist),    -   Muscarinic receptor (agonist),    -   Muscarinic M1 receptor (agonist),    -   Muscarinic M2 receptor (agonist),    -   Muscarinic M3 receptor (agonist),    -   Muscarinic M4 receptor (agonist),    -   Muscarinic M5 receptor (agonist)    -   Muscarinic receptor (partial agonist)    -   Adrenoceptor (agonist),    -   Alpha adrenoceptor (agonist),    -   Alpha 1 adrenoceptor (agonist),    -   Alpha 1A adrenoceptor (agonist),    -   Alpha 1B adrenoceptor (agonist)    -   Alpha 1D adrenoceptor (agonist)    -   Alpha 2 adrenoceptor (agonist),    -   Alpha 2A adrenoceptor (agonist),    -   Alpha 2B adrenoceptor (agonist),    -   Alpha 2C adrenoceptor (agonist),    -   Alpha 2 adrenoceptor (partial agonist)    -   Alpha 3 adrenoceptor (agonist),    -   Beta adrenoceptor (agonist),    -   Beta 1 adrenoceptor (agonist),    -   Beta 2 adrenoceptor (agonist),    -   Beta 3 adrenoceptor (agonist),    -   Dopamine receptor (agonist),    -   Dopamine D5 receptor (agonist)    -   Dopamine D1 receptor (agonist),    -   Dopamine D2 receptor (agonist),    -   Dopamine D3 receptor (agonist),    -   Dopamine D4 receptor (agonist),    -   Histamine receptor (agonist),    -   Histamine H1 receptor (agonist),    -   Histamine H2 receptor (agonist),    -   Histamine H3 receptor (agonist),    -   Histamine H4 receptor (agonist),    -   5-HT GPCR (agonist),    -   5-HT 1 (agonist),    -   5-HT 2 (agonist),    -   5-HT 4 (agonist),    -   5-HT 5a (agonist),    -   5-HT 5b (agonist)    -   5-HT 6 (agonist),    -   5-HT 7 (agonist),    -   Trace amine-associated receptor (agonist),    -   Trace amine-associated receptor-1 (agonist),    -   Trace amine-associated receptor-2 (agonist)    -   Trace amine-associated receptor-3 (agonist)    -   Trace amine-associated receptor-4 (agonist)    -   Trace amine-associated receptor-5 (agonist)    -   Trace amine-associated receptor-6 (agonist)    -   Trace amine-associated receptor-7 (agonist)    -   Trace amine-associated receptor-8 (agonist)    -   Trace amine-associated receptor-9 (agonist)    -   Apelin receptor (agonist),    -   Cannabinoid receptor (agonist),    -   Cannabinoid CB1 receptor (agonist),    -   Cannabinoid CB2 receptor (agonist),    -   Lysosphingolipid receptor (agonist),    -   Sphingosine-1-phosphate receptor-1 (agonist),    -   Lysophosphatidate-1 receptor (agonist)    -   Sphingosine-1-phosphate receptor-3 (agonist),    -   Lysophosphatidate-2 receptor (agonist)    -   Sphingosine-1-phosphate receptor-2 (agonist)    -   Sphingosine-1-phosphate receptor-4 (agonist),    -   Lysophosphatidate-3 receptor (agonist)    -   Sphingosine-1-phosphate receptor-5 (agonist)    -   Class A hormone protein GPCR (agonist),    -   FSH (agonist),    -   Luteinizing hormone receptor (agonist),    -   TSH (agonist),    -   Leukotriene (agonist),    -   Leukotriene BLT receptor (agonist),    -   Cysteinyl leukotriene receptor (agonist),    -   Melatonin (agonist),    -   Melatonin MT1 (agonist),    -   Melatonin MT2 (agonist),    -   Melatonin MT3 (agonist)    -   Class A nucleotide like GPCR (agonist),    -   Adenosine receptor (agonist),    -   P2Y receptor (agonist),    -   Class A orphan GPCR (agonist),    -   Ghrelin (agonist),    -   Class A peptide GPCR (agonist),    -   Angiotensin receptor (agonist),    -   Angiotensin I receptor (agonist),    -   Angiotensin II receptor (agonist),    -   Bombesin receptor (agonist),    -   Bombesin BB1 receptor (agonist)    -   Bombesin BB2 receptor (agonist)    -   Bombesin bb3 receptor (agonist),    -   Gastrin releasing peptide ligand,    -   Neuromedin B ligand    -   Neuromedin C ligand    -   Bradykinin receptor (agonist),    -   Bradykinin B1 receptor (agonist),    -   Bradykinin B2 receptor (agonist),    -   C3a receptor (agonist),    -   C5a (agonist),    -   CCK receptor (agonist),    -   CCK 1 receptor (agonist),    -   CCK 2 receptor (agonist),    -   Gastrin (agonist),    -   Chemokine (agonist),    -   CC chemokine receptor (agonist),    -   CCR1 chemokine (agonist),    -   CCR2 chemokine (agonist),    -   CCR3 chemokine (agonist),    -   CCR4 chemokine (agonist),    -   CCR5 chemokine (agonist),    -   CCR6 chemokine (agonist),    -   CCR7 chemokine (agonist)    -   CCR8 chemokine (agonist),    -   CCR9 chemokine (agonist)    -   CCR10 chemokine (agonist),    -   CCR11 chemokine (agonist)    -   CX3C chemokine receptor (agonist),    -   CX3CR1 chemokine (agonist),    -   XCR1 chemokine (agonist)    -   CXC chemokine receptor (agonist),    -   CXCR1 chemokine (agonist)    -   CXCR3 chemokine (agonist),    -   CXCR4 chemokine (agonist),    -   CXCR5 chemokine (agonist)    -   Adrenomedullin receptor (agonist),    -   Endothelin (agonist),    -   Endothelin ET-A (agonist),    -   Endothelin ET-B (agonist),    -   Galanin (agonist),    -   Galanin GAL1 (agonist),    -   Galanin GAL2 (agonist),    -   Galanin GAL3 (agonist)    -   IL-9 (agonist),    -   KiSS-1 receptor (agonist),    -   Melanin concentrating hormone (agonist),    -   MCH receptor-1 (agonist)    -   MCH receptor-2 (agonist)    -   Melanocortin (agonist),    -   Melanocortin MC1 (agonist),    -   ACTH receptor (agonist),    -   Melanocortin MC3 (agonist),    -   Melanocortin MC4 (agonist),    -   Melanocortin MC5 (agonist),    -   NK (agonist),    -   NK1 (agonist),    -   NK2 (agonist)    -   NK3 (agonist), Drugs: 1    -   Neuropeptide Y receptor (agonist),    -   Neuropeptide Y1 receptor (agonist)    -   Neuropeptide Y2 receptor (agonist),    -   Neuropeptide Y4 receptor (agonist),    -   Neuropeptide Y5 receptor (agonist),    -   Neuropeptide Y6 receptor (agonist)    -   Neurotensin receptor (agonist),    -   Neurotensin NTS1 (agonist),    -   Neurotensin NTS2 (agonist)    -   Orexin & neuropeptide FF receptor (agonist),    -   Orexin (agonist),    -   Opioid (agonist),    -   Delta opioid (agonist),    -   Kappa opioid (agonist),    -   Mu opioid (agonist),    -   ORL1 receptor (agonist),    -   Opioid (partial agonist)    -   Sigma opioid (agonist),    -   Orexin & neuropeptide FF receptor (agonist),    -   Neuropeptide FF receptor (agonist),    -   Neuropeptide FF1 receptor (agonist)    -   Neuropeptide FF2 receptor (agonist),    -   Orexin (agonist),    -   Orexin-1 (agonist)    -   Orexin-2 (agonist)    -   Protease-activated receptor (agonist),    -   Protease-activated receptor-1 (agonist),    -   Protease-activated receptor-2 (agonist),    -   Protease-activated receptor-3 (agonist)    -   Protease-activated receptor-4 (agonist)    -   Prokineticin receptor (agonist),    -   Prokineticin receptor-1 (agonist),    -   Prokineticin receptor-2 (agonist),    -   Somatostatin (agonist),    -   Somatostatin 1 (agonist),    -   Somatostatin 2 (agonist),    -   Somatostatin 3 (agonist),    -   Somatostatin 4 (agonist),    -   Somatostatin 5 (agonist),    -   Urotensin II (agonist),    -   Vasopressin like receptor (agonist),    -   Oxytocin (agonist),    -   Vasopressin (agonist),    -   Vasopressin V1 (agonist),    -   Vasopressin V2 (agonist),    -   Prostanoid receptor (agonist),    -   DP prostanoid (agonist),    -   PGD2 (agonist),    -   EP1 prostanoid (agonist),    -   PGE2 (agonist),    -   EP2 prostanoid (agonist),    -   PGE2 (agonist),    -   EP3 prostanoid (agonist),    -   PGE2 (agonist),    -   EP4 prostanoid (agonist),    -   PGE2 (agonist),    -   FP prostanoid (agonist),    -   PGF2 alpha (agonist),    -   IP prostanoid (agonist),    -   Prostacyclin (agonist),    -   Prostanoid receptor (partial agonist)    -   TP prostanoid (agonist),    -   Thromboxane A2 (agonist)    -   Succinate receptor 1 (agonist)    -   TRH (agonist),    -   TRH1 (agonist)    -   TRH2 (agonist)    -   Vomeronasal type-1 receptor (agonist)    -   Vomeronasal type-1 receptor-1 (agonist)    -   Vomeronasal type-1 receptor-2 (agonist)    -   Vomeronasal type-1 receptor-3 (agonist)    -   Vomeronasal type-1 receptor-4 (agonist)    -   Vomeronasal type-1 receptor-5 (agonist)    -   Apelin receptor (modulator),    -   Cannabinoid receptor (modulator),    -   Chemokine receptor-like 1 (modulator),    -   Lysosphingolipid receptor (modulator),    -   Class A hormone protein GPCR (modulator),    -   Leukotriene receptor (modulator),    -   Melatonin receptor (modulator),    -   Class A nucleotide like GPCR (modulator),    -   Class A orphan GPCR (modulator),    -   PAF receptor (modulator),    -   Class A peptide GPCR (modulator),    -   Prostanoid receptor (modulator),    -   Succinate receptor 1 (modulator)    -   TRH receptor (modulator),    -   Vomeronasal type-1 receptor (modulator),        Class B GPCRs    -   G-protein coupled receptor-3 (modulator),    -   G-protein coupled receptor-3 (agonist)    -   G-protein coupled receptor-3 (antagonist),    -   G-protein coupled receptor-6 (modulator),    -   G-protein coupled receptor-6 (agonist)    -   G-protein coupled receptor-6 (antagonist),    -   G-protein coupled receptor-12 (modulator),    -   G-protein coupled receptor-12 (agonist)    -   G-protein coupled receptor-12 (antagonist),    -   G-protein coupled receptor-14 (modulator)    -   G-protein coupled receptor-14 (agonist)    -   G-protein coupled receptor-14 (antagonist)    -   Class B GPCR (agonist),    -   CRF-1 receptor (agonist)    -   CRF-2 receptor (agonist),    -   Calcitonin receptor (modulator),    -   Calcitonin (agonist),    -   Calcitonin (antagonist),    -   ACTH releasing factor receptor (modulator),    -   CRF-1 receptor (modulator),    -   CRF-1 receptor (agonist)    -   CRF-1 receptor (antagonist),    -   CRF-2 receptor (modulator),    -   CRF-2 receptor (agonist),    -   CRF-2 receptor (antagonist),    -   ACTH releasing factor (agonist),    -   CRF-1 receptor (agonist)    -   CRF-2 receptor (agonist),    -   ACTH releasing factor (antagonist),    -   CRF-1 receptor (antagonist),    -   CRF-2 receptor (antagonist),    -   Glucagon-like peptide receptor (modulator),    -   Glucagon-like peptide 1 receptor (modulator),    -   Glucagon-like peptide 2 receptor (modulator),    -   Glucagon-like peptide (agonist),    -   Glucagon-like peptide (antagonist),    -   Glucagon receptor (modulator),    -   Glucagon (agonist),    -   Glucagon (antagonist),    -   GHRH receptor (modulator),    -   GHRH (agonist),    -   Growth hormone releasing factor (antagonist),    -   PACAP type I receptor (modulator),    -   PACAP type I receptor (agonist),    -   PACAP type I receptor (antagonist)    -   PTH receptor (modulator),    -   PTH-1 receptor (modulator)    -   PTH-2 receptor (modulator)    -   PTH (agonist),    -   PTH (antagonist),    -   Secretin receptor (modulator),    -   Secretin (agonist),    -   Secretin (antagonist)    -   VIP receptor (modulator),    -   VIP-1 receptor (modulator),    -   VIP-2 receptor (modulator),    -   VIP (agonist),    -   VIP (antagonist),        Class C GPCRs

Class C GPCR (modulator),

-   -   Class C GPCR (agonist),    -   GABA B receptor (agonist),    -   Metabotropic glutamate receptor (agonist),    -   Metabotropic glutamate receptor 1 (agonist),    -   Metabotropic glutamate receptor 2 (agonist),    -   Metabotropic glutamate receptor 3 (agonist),    -   Metabotropic glutamate receptor 4 (agonist),    -   Metabotropic glutamate receptor 5 (agonist),    -   Metabotropic glutamate receptor 6 (agonist)    -   Metabotropic glutamate receptor 7 (agonist)    -   Metabotropic glutamate receptor 8 (agonist)

Preferably, said cell-associated antigen is a membrane-spanning antigen,including but not limited to an antigen selected from ion channels suchas e.g. P2X7.

The skilled person will appreciate that there may be different specificthree dimensional conformations that are encompassed by the term“natural conformation”. If, for example, a protein has two or moredifferent conformations whilst being in a membrane environment, allthese conformations will be considered “natural conformations”. This isexemplified by receptors changing their conformation by activation, e.g.the different activation states of rhodopsin induced by light, or ionchannels showing a “closed” or “open” conformation. The inventionencompasses immunoglobulin sequences to any one of these differentnatural conformations, i.e. to the different kinds of conformationalepitopes that may be present.

A “nucleic acid” of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one embodiment of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the cell associatedantigen or immunoglobulin sequences of the invention, and/or can beisolated from a suitable natural source. To provide analogs; nucleotidesequences encoding naturally occurring V_(HH) domains can for example besubjected to site-directed mutagenesis, so at to provide a nucleic acidof the invention encoding said analog. Also, as will be clear to theskilled person, to prepare a nucleic acid of the invention, also severalnucleotide sequences; such as at least one nucleotide sequence encodinga Nanobody and for example nucleic acids encoding one or more linkerscan be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring GPCR as a template. These and other techniques willbe clear to the skilled person, and reference is again made to thestandard handbooks, such as Sambrook et al. and Ausubel et al.,mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism, or in a form suitable for genetic immunization. Forinstance, the genetic constructs of the invention may be in the form ofa vector, such as for example a plasmid, cosmid, YAC, a viral vector ortransposon. In particular, the vector may be an expression vector, i.e.a vector that can provide for expression in vitro and/or in vivo (e.g.in a suitable host cell, host organism and/or expression system).

In a preferred but non-limiting embodiment, a genetic construct of theinvention comprises

-   a) at least one nucleic acid of the invention; operably connected to-   b) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   c) one or more further elements of genetic constructs known per se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art (as    further described herein); and in which said “further elements”    present in the genetic constructs may for example be 3′- or 5′-UTR    sequences, leader sequences, selection markers, expression    markers/reporter genes, and/or elements that may facilitate or    increase (the efficiency of) transformation or integration. These    and other suitable elements for such genetic constructs will be    clear to the skilled person, and may for instance depend upon the    type of construct used, the intended host cell, host organism or    animal to be immunized; the manner in which the nucleotide sequences    of the invention of interest are to be expressed (e.g. via    constitutive, transient or inducible expression); and/or the    transformation/vaccination technique to be used. For example,    regulatory sequences, promoters and terminators known per se for the    expression and production of antibodies and antibody fragments    (including but not limited to (single) domain antibodies and ScFv    fragments) may be used in an essentially analogous manner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promoter). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

For some (further) non-limiting examples of the promoters, selectionmarkers, leader sequences, expression markers and further elements thatmay be present/used in the genetic constructs of the invention—such asterminators, transcriptional and/or translational enhancers and/orintegration factors—reference is made to the general handbooks such asSambrook et al. and Ausubel et al. mentioned above, as well as to theexamples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat.No. 6,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other exampleswill be clear to the skilled person. Reference is also made to thegeneral background art cited above and the further references citedherein.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the Nanobody or polypeptide of theinvention, or for genetic vaccination. Suitable hosts or host cells willbe clear to the skilled person, and may for example be any suitablefungal, prokaryotic or eukaryotic cell or cell line or any suitablefungal, prokaryotic or eukaryotic organism.

According to one non-limiting embodiment of the invention, theimmunoglobulin sequences, Nanobody or polypeptide of the invention isglycosylated. According to another non-limiting embodiment of theinvention, the immunoglobulin sequences, Nanobody or polypeptide of theinvention is non-glycosylated.

In the context of the present invention, “genetic vaccination” includesany known methods or means to transfer a nucleic acid sequence, e.g. aDNA sequence, into a target animal that is suitable for inducing animmune response to a protein encoded by said nucleic acid sequence. Theskilled person knows standard ways of genetic vaccination. According tothe invention, genetic vaccination can be performed by a needle-free jetinjection, by a ballistic method, by needle-mediated injections such astattoo, by topical application of the DNA onto the skin in patches or byany of these administration methods followed by in vivo electroporation,and furthermore includes vaccination performed by intradermal,intramuscular or subcutaneous administration of DNA.

In the context of genetic vaccination, the term “genetic” refers to anysuitable type or kind of nucleic acid molecule, e.g. as defined herein,such as DNA, RNA, cDNA, double stranded DNA, including nucleic acidmolecules comprising modified nucleotides, such as PNA, wherein saidnucleic acid molecule encodes the cell associated antigen as definedherein, and is suitable for causing expression in the non-human animalsuch that an immune response can be generated.

Examples of nucleic acid molecules comprise DNA, RNA, PNA, cDNA, doublestranded DNA, as well as other forms of nucleic acid moleculescomprising chemical modifications to increase e.g. stability in vivo orin vitro.

The nucleic acid molecules can be in the form of an expression vector,plasmid, or any other nucleic acid molecule or genetic constructsuitable for expressing the antigen in the animal. Suitable nucleic acidmolecules are known to the skilled person, and include commerciallyavailable expression vectors and plasmids. Specific, non-limitingexample include the commercially available pVAX1 construct, or theeukaryotic expression vector pRc/CMV-Hbs(s) encoding the Hepatitis Bsmall surface antigen (HBSAg) obtainable from Aldevron, which can beengineered by routine means to express the antigen of interest.

Some non-limiting examples of vectors for use in mammalian cellsinclude: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMC1neo (Stratagene),pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo(ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC37565), as well as viral-based expression systems, such as those basedon adenovirus;

Genetic vaccination will be performed by a suitable nucleic acid orgenetic construct, comprising elements suitable for expressing thetarget antigen in the non-human animal. Such elements will compriseelements that encode the structural information of the antigen, or partsthereof, provided that the conformational epitopes of interest arerepresented by this structural information. The genetic construct mayalso comprise elements that are responsible for the control ofexpression, such as suitable promoters, enhancers, terminators and othercontrol sequences known to the skilled person. Specifically, theinvention encompasses the use of promoters allowing constitutiveexpression after in vivo transfection. A specific, non-limiting exampleof a suitable promoter is the constitutive Cytomegalovirus (CMV)promoter. The skilled person knows a multitude of further suitablepromoters, including, but not limited to promoters for expression inmammalian cells: human cytomegalovirus (hCMV) immediate earlyenhancer/promoter; human cytomegalovirus (hCMV) immediate early promotervariant that contains two tetracycline operator sequences such that thepromoter can be regulated by the Tet repressor; Herpes Simplex Virusthymidine kinase (TK) promoter; Rous Sarcoma Virus long terminal repeat(RSV LTR) enhancer/promoter; elongation factor 1α (hEF-1α) promoter fromhuman, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1 longterminal repeat promoter; β-actin promoter.

The invention encompasses production of the nucleic acid moleculesrequired for genetic vaccination in suitable quantities by methods knownin the art. For example, endotoxin-low plasmid DNA can be produced usingendotoxin-free Gigaprep kit (Qiagen) according to the manufacturer'sinstructions.

For genetic vaccination, the nucleic acid molecule as described hereinmay be formulated in a suitable fashion. For example, the nucleic acidmolecule, such as a vector or plasmid is finally reconstituted inendotoxin-free H₂O, preferably LAL (limulus amaebocyte lysate) H₂O (i.e.water that has been tested for endotoxin by LAL) or in endotoxin-free0.9% NaCl in LAL H₂O, or another suitable buffer or solution known tothe skilled person. The reconstituted nucleic acid molecule can bestored in solution in aliquots at −20° C., or alternatively can bestored in lyophilized form for reconstitution prior to use.

The nucleic acid molecule will be diluted to a suitable dilution for usein genetic vaccination, e.g. at a concentration of 0.1 to 10 mg/ml,specifically 1 to 5 mg/ml, more specifically 1 mg/mL.

For genetic vaccination, the nucleic acid molecule will be administeredto the animal in a suitable fashion, as outlined herein. Specificexamples of suitable methods for intradermal application of DNA compriseneedle-free jet injection (Pig-jet), a tattoo method (Bins, et al.,Nature Medicine 11:899-904), needle-free jet injection using theVacci-jet (Robbins Instruments, USA), topical administration of DNA ontothe skin via patches or the Helios Gene-gun (Biorad) as ballistic methodto administer the DNA. All DNA administration methods can be followed byin vivo electroporation to enhance cellular transfection efficiency.

In the context of any of the above methods, the nucleic acid may beassociated with a suitable carrier. For example, the nucleic acid may beassociated with particles, including, but not limited to gold particles.The skilled person knows routine techniques for associating nucleicacids with suitable carriers.

In the context of the present invention, “non-human animal” includes,but is not limited to vertebrate, shark, mammal, lizard, camelid, llama,preferably camelids and most preferably llama or alpaca.

B) THE METHOD OF THE PRESENT INVENTION

The present invention relates to a method for the generation ofimmunoglobulin sequences that can bind to and/or have affinity for acell-associated antigen, as defined herein. The method comprises, but isnot limited, to the following steps:

a) genetic vaccination of a non-human animal with a nucleic acidencoding said cell-associated antigen or a domain or specific part ofsaid cell associated antigen or a domain or specific part of said cellassociated antigen as a genetic fusion to for example immune modulatorygenetic elements or a domain or specific part of said cell associatedantigen grafted on a camelid orthologue sequence; andb) optionally boosting the animal with said antigen in its naturalconformation selected from cells comprising natural or transfected cellsexpressing the cell-associated antigen (or antigen domains or a specificpart of said antigen), cell derived membrane extracts; vesicles or anyother membrane derivative harbouring enriched antigen, liposomes, orvirus particles expressing the cell associated antigenc) screening a set, collection or library of immunoglobulin sequencesderived from said non-human animal for immunoglobulin sequences that canbind to and/or have affinity for said cell-associated antigen

Thus, in general terms the method of the present invention includesgenetic vaccination of a non-human animal as defined herein. In oneparticular embodiment, the non-human animal is a camelid.

On particular advantage of the present invention resides in the factthat it provides a robust method for generating immunoglobulin sequencesthat is widely applicable to a range of antigens. The method of theinvention is not limited by the accessibility of protein antigen. Inparticular, there is no requirement for purified antigen.Advantageously, the method also excludes the need for antigen expressedon a cell, or present in a membrane environment, in that it can beperformed by solely using genetic vaccination. Hence, the method of thepresent invention is broadly applicable to any of the antigensexemplified above, but not limited thereto. In particular, the presentmethod is applicable to antigens for which a corresponding nucleic acidsequence is known, or can be identified by routine means.

Hence, the present invention is advantageous as compared to prior artmethods that lack such robust and broad applicability. In particularthere is no teaching in the art for such a robust method for thegeneration of immunoglobulin sequences in animals such as camelids, inparticular llama.

Specifically; the present invention provides an improved method forgenerating immunoglobulin sequences against cell-associated antigens,which, according to one specific embodiment, is without the need for aboost with purified protein, by inducing an immune response via DNAvaccination and subsequent screening for immunoglobulin sequences thatcan bind the cell-associated antigen. More particularly, the presentinvention provides a method for the generation of immunoglobulinsequences, including Nanobodies, against a cell-associated antigencomprising the steps of:

a) Vaccination of a camelid with a nucleic acid encoding saidcell-associated antigen (or antigen domains or a specific part of saidantigen); and

b) Screening a set, collection or library of immunoglobulin sequencesderived from said camelid for immunoglobulin sequences that can bind toand/or have affinity for said cell-associated antigen.

It has also been surprisingly found, that even in cases where the serumantibody titre was lower after DNA vaccination as compared to protein orcell based immunization, the screening for specific immunoglobulinsequences provided comparable hit rates, i.e. a comparable frequency ofspecific immunoglobulin sequences could be obtained. Moreover, theaffinity of the identified binders was high (as defined herein). Thisunderlines the particular advantage of the present invention ofresulting in a more specific high affinity immunoglobulin response, andallowing for more efficient screening and isolation of specificimmunoglobulin sequences. It was unforeseeable from the prior art thatsuch advantages can be obtained by DNA vaccination, in particular whenthe immunized animal exhibits a lower immune response in terms of serumantibody titre.

In an alternative embodiment, the present invention provides a methodfor the generation of immunoglobulin sequences, including Nanobodies,against a cell-associated antigen comprising the steps of:

a) Vaccination of a camelid with a nucleic acid encoding saidcell-associated antigen (or antigen domains or a specific part of saidantigen);

b) boosting the camelid with cell associated antigen in its naturalconformation, e.g. by use of transfected cells expressing thecell-associated antigen (or antigen domains or a specific part of saidantigen), with cell membrane extracts or with virus particles expressingthe cell associated antigen, andc) Screening a set, collection or library of immunoglobulin sequencesderived from said camelid for immunoglobulin sequences that can bind toand/or have affinity for said cell-associated antigen.Vaccination

In the method of the invention, genetic vaccination suitable forinducing an immune response in the animal is performed. Morespecifically, the genetic vaccination must be suitable to induce animmune response as reflected in the generation of immunoglobulinsequences in the animal. The detection of an antibody response in theserum of the animal is also referred to as “serum conversion”. Theskilled person can monitor genetic vaccination by determining theantibody response by routine means. Thus, the skilled person can readilydetermine the adequate dosage and frequency that is required forinducing an appropriate antibody response.

Preferably, the genetic vaccination will induce an adequate antibodytitre. The antibody titre will correspond to the number of specificantibody producing cells, which will allow the generation ofimmunoglobulin sequences by isolation and/or screening. However, aspointed out above, the method of the present invention allows for thesuccessful isolation of high affinity immunoglobulin sequences even whenthere is only a low serum antibody titre as compared to conventionalmethods. For example, genetic vaccination will allow the successfulisolation of high affinity immunoglobulin sequences at serum antibodytitres that are e.g. 3 fold lower as compared to the serum titresobtained after a protein boost (which are comparable to antibody tiresobtainable by conventional protein immunization techniques). In aparticular embodiment, the serum antibody titres may be 5 fold lower,preferably 10 fold lower. Serum titres can be determined by conventionalmethods, including e.g. ELISA or FACS.

A further aspect of importance for the present invention is the breadthof the antibody repertoire obtained by genetic vaccination. Inparticular, it is one aspect of the present invention that the antibodyresponse is directed to both linear and conformational epitopes, andimportantly is directed to membrane dependent conformational epitopes.

Thus, the present invention relates to genetic vaccination suitable forobtaining an antibody response of an adequate titre and breadth in thenon-human animals.

In one embodiment, the present invention may involve a single geneticvaccination at one or multiple sites of the animal. For example, acamelid may be injected in 1, 2, 3, 4, 5 or multiple sites, that may beadjacent to each other or distributed over the body of the animal insuitable locations. In a specific example, the camelid, e.g. a llama,receives genetic vaccination on up to five adjacent sites on the neck.It is self evident that the areas for genetic vaccination have to beclean and free of hair. Hence, the invention encompasses suitable meansof removing hair, such as shaving and chemical means such as depilationcreams or physical removal of hairs via tape.

In one aspect of the invention it has surprisingly been found that thelocation of administering the DNA vaccine has an influence on theobtainable immunoglobulin sequences, in particular in terms of diversityand epitope preference.

The invention encompasses repeated genetic vaccination, e.g. 2, 3, 4 or5 sequences of genetic vaccination in suitable time intervals. Suchintervals will be preferably days to weeks, e.g. 3 days to 4 weeks, morepreferably 5 days to two weeks. Suitable intervals comprise, geneticvaccination on 0, 3, 7, 21, 24, 28, 56, 59 and 63 days, alternatively on0, 14, 28 and 57 days, alternatively at days 0, 3, 7, 21, 24, 28, 56, 59and 63, alternatively on days 0, 14, 28 and 42.

In one specific embodiment, genetic vaccination is performed on a weeklyor every other week, until an adequate antibody response is elicited inthe animal.

In a further specific embodiment, intradermal administration, e.g. byneedle free injection, is performed on days 0, 14, 28 and 57. In anotherspecific embodiment, the short-interval tattoo method, tattooing isperformed at days 0, 3, 7, 21, 24, 28, 56, 59 and 63. A further specificexample comprises immunization by needle free injection on days 0, 14,28 and 42. In a ballistic method of genetic vaccination, the dose may be12 shots of 1 μg DNA/mg gold at a pressure setting of up to 500-600 psi,administered at intervals of 0, 14, 28 and 42 days.

In one particular embodiment, the present invention relates to geneticvaccination using a suitable DNA administration technique followed by invivo electroporation. It has been surprisingly found that this mode ofadministration is advantageous as compared to the conventional methodsof genetic vaccination. For example, in vivo electroporation isadvantageous in terms of vaccination efficacy, i.e. it results in a morepronounced, and/or more reliable immune response. More reliable in thiscontext means that a lower variability in the immune response, an inparticular in the number of “hits” obtainable by screening, betweenindividual animals is observed. Moreover, the use of electroporationallows, by changing the settings of the system, to readily adapt thevaccination protocol to the required penetration depth, e.g. to selectbetween intradermal, subcutaneous or intramuscular vaccination.Moreover, considering the relatively thick and tough skin of someanimals, such as camelids, electroporation also allows for a goodvaccination efficacy, and the ready adaptation to various differentlocations of immunization characterized by different skin properties.

The skilled person can select a suitable dose of nucleic acid moleculesfor genetic vaccination. For example, 0.1-10 mg nucleic acid,specifically 1-5 mg, more specifically 1 to 2 mg, or 1 mg nucleic acidcan be used for one application of genetic vaccination (e.g. on day 0),resulting in a cumulative dose that depends on the number of repeatgenetic vaccinations.

When a suitable antibody response has been confirmed in the animal,immunoglobulin sequences can in one embodiment of the invention bedirectly isolated from said animal, i.e. without protein boost, bymethods as described herein. Detection of antibody responses can be doneby routine means, such as ELISA, RIA, FACS, or any other method fordetecting antibodies.

Protein Boost

Alternatively, the method also includes boosting the animal with asuitable source of protein. In particular it is envisaged to boost theanimal with compositions that comprise the cell associated antigen asdefined herein, in particular a transmembrane antigen, in its naturalconformation. Such compositions may comprise cells expressing theantigen, or fragments or derivatives of the cell, such as membranefractions, isolated organelles, or other suitable preparations. Alsoenvisaged are viruses, liposomes, micelles or other systems that aresuitable for containing the cell associated antigen in its naturalconformation.

In one aspect of the invention the antigen can be expressed on ahomologous cell. For example, for immunization of a camelid, the antigencan be expressed on a camelid cell. The camelid immune system will betolerant to the camelid cell, i.e. it will not mount an immune responseto most of the antigens comprised in this cell. However, if aheterologous antigen, including but not limited to cell associatedantigens as defined herein, is artificially introduced into said cell,the immune system of the animal will mount an immune responsespecifically directed to said antigen. This has the advantage that theimmune response will be mainly directed to the antigen of interest, i.e.it will be characterized by an enhanced specificity towards thisantigen. The skilled person will appreciate that this approach can beused for related species. For example, camel derived cells can be usedfor immunization of llama, and vice versa, in view of their closerelationship.

Any suitable cell that is homologous to the animal to be immunized canbe used. For example, camelid cells can be used for immunization ofcamelids, e.g. llama cells for immunization of llama. Suitable cellswill comprise, but are not limited to, cells that are spontaneouslyimmortal, e.g. cancer cells or undifferentiated cells, such asembryo-derived cells. Suitable cells also encompass cells immortalizedartificially by known means.

Cells can advantageously be treated prior to administration to theanimals; such that their proliferation in vivo is reduced or eliminated.Suitable treatments comprise, but are not limited to chemical andphysical treatments. One specific example of a suitable physicaltreatment is irradiation with X rays such that the cells can no longerproliferate.

Any of the above cells can also be used for immunizing a non-humananimal as defined herein in its own right, i.e. independent of DNAvaccination.

Preferably the protein is enriched in any of the above preparations, inorder to strengthen the immune response. For example, recombinantexpression in cells using highly efficient promoters can be used toincrease the quantity of antigen per cell. In one embodiment, when usingcamelids as the non-human animal, the cells expressing the antigen ofinterest can be camelid derived cells, preferably immortalized camelidderived cells. The cells will be genetically modified to express thesaid antigen.

Moreover, the skilled person will understand that the invention alsoencompasses the use of an adjuvant commonly used in order to enhance animmune response in the context of vaccination. The protein preparationmay also be in a physical form that enhances the immune response, suchas e.g. a gel or emulsion. Specific, non-limiting examples of anadjuvant include Stimune or Specol (CEDI Diagnostics, Lelystad, TheNetherlands), Freund's Complete Adjuvant, Freund's Incomplete Adjuvant,TiterMax (Gold), monophosphoryl lipid A (MPL), Alum, QuilA, CpG DNA.

The present invention comprises a single or multiple boosts with thesaid source of protein in its natural conformation (optionally using anadjuvant). The protein boosts will be performed at suitable intervals,which can be determined by routine means, e.g. by monitoring theimmunoglobulin response in the animals.

The boost can be performed by different routes of administration,including, but not limited to, intradermal, subcutaneous, orintramuscular administration.

In one particular embodiment the present invention relates to thereduction of the number of protein administrations required in an animalto elicit a suitable immune response. Thus, the geneticvaccination-protein boost (also referred to as “prime-boost”) strategywill eliminate the need for repeated protein boost of the animal. This,for one, reduces the burden on the animal, facilitates and speeds up theprocedure, and reduces the amount of antigen that is necessary forraising the immunoglobulin sequences. Thus, the geneticvaccination-protein boost strategy of the present invention cansurprisingly result in the same antibody titres in the blood of ananimal, as a conventional method comprising multiple protein boosts,even without or if only a single protein boost is given after geneticvaccination.

A further particular advantage of a prime-boost strategy using cells asantigen source for the boost resides in the fact that the antibodyresponse will be characterized by a particularly high specificity ascompared to known approaches. In other words, because of the DNApriming, the recall immune response elicited by the cell boost willprimarily be directed to the antigen of interest, and any otherantigenic determinants on the cells will not significantly affect theoverall immune response. Thus, the DNA prime-cell boost according to thepresent invention is particularly advantageous in terms of specificity,hence resulting in an advantageous “hit rate”, i.e. a large number ofspecific immunoglobulin sequences upon screening.

Thus, in the embodiment comprising a protein boost, particular technicaleffects comprise the enhanced immune response. Moreover, the sequencediversity of different functional Nanobodies belonging to the sameB-cell lineage will be enhanced. The boost according to the inventioncauses introduction of formerly unidentified amino acid substitutionscompared with sequences identified after the genetic immunization only,which is an indication for boost mediated in vivo maturation.

Screening/Isolating Immunoglobulin Sequences

The genetic vaccination as described herein will induce an immuneresponse in the animal. Then, a set, collection or library ofimmunoglobulin sequences is isolated from the animals. “Isolation”includes a) the separation of sequences from the animal, e.g. bysampling suitable tissues, and b) the singling out of specific sequencese.g. by screening, i.e. the isolation of “hits” of specific binders.

The skilled person is well acquainted with techniques for establishingsuitable sets, collection or libraries of immunoglobulin sequences, andscreening thereof for the sequences of interest. The skilled person canmake general reference to the techniques described in for example WO02/085945 and in WO 04/049794. Reference can also be made to techniquesand methods described in WO 99/37681, WO 01/90190, WO 03/025020 and WO03/035694. Alternatively, improved synthetic or semi-synthetic librariesderived from e.g. V_(HH) libraries, obtained form the animals immunizedin accordance with the present invention, may be used, such as V_(HH)libraries obtained from V_(HH) libraries by techniques such as randommutagenesis and/or CDR shuffling, as for example described in WO00/43507.

The invention includes the isolation of material from the animal whichcomprises immunoglobulin sequences, such as, but not limited to,antibody producing cells. For example, peripheral blood monocytes(PBMCs) can be isolated by conventional means. Other material includesperipheral blood lymphocytes (PBLs), peripheral lymph nodes, inparticular lymph nodes draining the site of immunization, the spleen,bone marrow, or other immunologically relevant materials.

In one specific, non-limiting example, B-cell containing blood samplescan be collected, and peripheral blood lymphocytes (PBLs) can bepurified by standard methods. For example, a density gradientcentrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden)can be employed according to the manufacturer's instructions.

Any of the above described material, including e.g. PBLs isolated fromthe animal will comprise a multitude of immunoglobulin sequences, i.e. aset, collection or library of immunoglobulin sequences.

Amongst this multitude of immunoglobulin sequences, e.g. expressed onPBMCs, the desired immunoglobulin specificities can be directlyisolated, e.g. by immunopanning of the cells.

Alternatively, nucleic acid sequences coding for the set, collection orlibrary of immunoglobulin sequences can be isolated, transferred, andexpressed on a set, collection or sample of cells or viruses.

The genetic material can be isolated and processed further by suitablemeans to isolate such sequences that code for the immunoglobulinsequences of the desired specificity. To this end, e.g. the nucleic acidsequences encoding the said multiplicity of immunoglobulin sequences canbe extracted from the material by suitable means, and transferred into arecipient cell or virus for expression. The skilled person is familiarwith suitable techniques for extraction of immunoglobulin sequences andmanipulating these sequences for expression, e.g. in an expressionlibrary in cells or viruses. Some non-limiting examples comprise thegeneration of an expression library in e.g. E. coli or bacteriophages.

In one specific, non-limiting example, total RNA can be extracted fromthe said material. The total RNA can be converted into cDNA by knownmeans. Using this cDNA, immunoglobulin sequences, such as e.g. theNanobody repertoire, can be amplified by routine means, including e.g.PCR, or nested PCR methods as known in the art (see patent referencesabove).

Nucleic acid molecules comprising immunoglobulin sequences can bedigested by use of suitable restriction enzymes, optionally followed bypurification e.g. by gel electrophoresis. The digested sequences can beligated into corresponding restriction sites in a suitable geneticconstruct, such as a vector or plasmid. Non-limiting examples ofsuitable vectors include phage display vectors, e.g. pAX50, pAX50contains the LacZ promoter, a coliphage pill protein coding sequence, aresistance gene for ampicillin or carbenicillin, a multicloning site(harboring the SfiI and BstEII restriction sites) and a chimeric leadersequence consisting of gene3 and Erwinia carotovora pelB motifs. Thisdisplay vector allows the production of phage particles, expressing theindividual Nanobodies as a fusion protein with the geneIII product.

The ligated nucleic acid molecule can be used to obtain a library, e.g.by transformation of a suitable host organism, like E. coli. The skilledperson knows suitable techniques of transformation, e.g. chemicalmethods, electroporation, and others. Thus, a library of a suitablesize, e.g. 1E7 to 1E8, can be obtained.

In one embodiment, libraries can be rescued by growing the bacteria tologarithmic phase (e.g. OD600=0.5), followed by infection with helperphage to obtain recombinant phage expressing the repertoire of clonedimmunoglobulin sequences on tip of the phage as a pill fusion protein,the obtained phage can be stored, e.g. after filter sterilization, forfurther use, e.g. at 4° C.

A set, collection or library of cells or viruses is screened for cellsor viruses that express immunoglobulin sequences that can bind to and/orhave affinity for said cell-associated antigen, more specifically, anucleic acid sequence that encodes the immunoglobulin sequence that canbind to and/or has affinity for said cell-associated antigen can bepurified and/or isolated from the cell or virus, followed by expressionof said immunoglobulin sequence.

Thus, the present invention also encompasses suitable screening step(s),to select and isolate the immunoglobulin sequences directed to theantigen of interest (or nucleic acid sequences encoding the same) from amultitude of sequences present in the non-human animal. Such screeningmethods encompass all methods that are suitable for singling out a cell,virus, expression construct, or sequence that relates to theimmunoglobulin sequence of interest. The skilled person is well aware ofa multitude of suitable techniques, including phage display,immunopanning, etc. Of course the invention also relates to combinationsof known methods. Suitable combinations will be apparent to the skilledperson.

In one specific embodiment, the library of phages expressingimmunoglobulin sequences can be selected by a single round, or multiplerounds of panning on a suitable source of cell-associated antigen,including, but not limited to (immobilized) cells or liposomescomprising the antigen of interest.

After a round of selection, e.g. by immunopanning, the output can berecloned as a pool into a suitable expression vector for furtherselection and/or processing.

According to the invention, the immunoglobulin sequence that can bind toand/or has affinity for said cell-associated antigen can be purifiedand/or isolated.

The skilled person can use standard techniques for production ofimmunoglobulins. Thus, after a cell, virus or nucleic acid sequenceencoding the immunoglobulin sequence of interest has been identified bya screening method, the said immunoglobulin sequence can be produced,e.g. by means of recombinant expression. For this purpose, the cell orvirus can be used directly, or the nucleic acid encoding theimmunoglobulin sequence can be transferred into a suitable expressionsystem, including a suitable host cell. Host cells include mammaliansystems, such as CHO cells, eukaryotic systems such as insect cells orfungi, including e.g. Pichia pastoris, and prokaryotic systems such asE. coli. The skilled person knows suitable expression vectors and toolsfor use in expressing immunoglobulin sequences in these host systems.

The immunoglobulin sequences, Nanobodies and nucleic acids of theinvention can be prepared in a manner known per se, as will be clear tothe skilled person from the description herein. The skilled person willunderstand which of the specific examples are suitable for geneticvaccination, for the generation and/or screening of sets, collections orlibraries of immunoglobulin sequences, or for the production ofimmunoglobulin sequences after selection of antigen specific sequences.

For example, the polypeptides of the invention can be prepared in anymanner known per se for the preparation of antibodies and in particularfor the preparation of antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments).

As will be clear to the skilled person, one particularly useful methodfor preparing a polypeptide of the invention generally comprises thesteps of:

the expression, in a suitable host cell or host organism (also referredto herein as a “host of the invention”) or in another suitableexpression system of a nucleic acid that encodes said Nanobody orpolypeptide of the invention (also referred to herein as a “nucleic acidof the invention”, this term is also used for the genetic constructs forvaccination, as will be apparent from the specific context), optionallyfollowed by:

isolating and/or purifying the Nanobody or polypeptide of the inventionthus obtained.

Moreover, the produced immunoglobulins can be purified by standardtechniques, including precipitation, affinity chromatography, sizeexclusion chromatography, ion exchange chromatography, HPLC, filtration,and other known purification methods.

Furthermore, the immunoglobulin sequences can be further characterizedby known methods, e.g. to determine their epitope specificity, bindingkinetics, etc.

Immunoglobulin Sequences

The invention also relates to immunoglobulin sequences, i.e. thepolypeptide molecules, obtainable by a method as described herein, andcompositions comprising the said immunoglobulin sequences. Suchcompositions comprise compositions for research purposes as well aspharmaceutical compositions for use in therapy. The skilled person isfamiliar with standard techniques and formulations for therapeuticapplication of immunoglobulin sequences. Thus, in one aspect the methodof the present invention encompasses the purification of specificimmunoglobulin sequences and their formulation as a pharmaceuticalcomposition.

The present invention provides immunoglobulin sequences in essentiallyisolated form, e.g. in a form that is at least 90% pure, at least 95%pure, at least 98%, at least 99%, or at least 99.99% pure. In onenon-limiting embodiment, purity means that no sequences of otherimmunoglobulins are present in the preparation. In a furthernon-limiting embodiment purity means that no contaminants from theproducing organism are present in the composition.

The present invention also encompasses immunoglobulin sequences that arederivatives of the immunoglobulin sequences obtainable by the methodsdisclosed herein. For example, the invention encompasses humanizedimmunoglobulin sequences obtainable by methods known in the art.Moreover, the invention encompasses camelized immunoglobulin sequences,also obtainable by methods known in the art. The invention alsoencompasses known structural variants of immunoglobulin sequences.

Immunoglobulin Sequences Directed Against Hepatitis B Small SurfaceAntigen

The present invention relates to immunoglobulin sequences that aredirected against (as defined herein) Hepatitis B small surface antigens,as well as to compounds or constructs, and in particular proteins andpolypeptides, that comprise or essentially consist of one or more suchimmunoglobulin sequences (also referred to herein as “immunoglobulinsequences of the invention”, “compounds of the invention”, and“polypeptides of the invention”, respectively). The invention alsorelates to nucleic acids encoding such immunoglobulin sequences andpolypeptides (also referred to herein as “nucleic acids of theinvention” or “nucleotide sequences of the invention”); to methods forpreparing such immunoglobulin sequences and polypeptides; to host cellsexpressing or capable of expressing such immunoglobulin sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such immunoglobulin sequences, polypeptides,nucleic acids and/or host cells; and to uses of such immunoglobulinsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Immunoglobulin Sequences Directed Against Ion Channels

The present invention relates to immunoglobulin sequences that aredirected against (as defined herein) ion channels, as well as tocompounds or constructs, and in particular proteins and polypeptides,that comprise or essentially consist of one or more such immunoglobulinsequences (also referred to herein as “immunoglobulin sequences of theinvention”, “compounds of the invention”, and “polypeptides of theinvention”, respectively). The invention also relates to nucleic acidsencoding such immunoglobulin sequences and polypeptides (also referredto herein as “nucleic acids of the invention” or “nucleotide sequencesof the invention”); to methods for preparing such immunoglobulinsequences and polypeptides; to host cells expressing or capable ofexpressing such immunoglobulin sequences or polypeptides; tocompositions, and in particular to pharmaceutical compositions, thatcomprise such immunoglobulin sequences, polypeptides, nucleic acidsand/or host cells; and to uses of such immunoglobulin sequences orpolypeptides, nucleic acids, host cells and/or compositions, inparticular for prophylactic, therapeutic or diagnostic purposes, such asthe prophylactic, therapeutic or diagnostic purposes mentioned herein.

Xu et al., Nature Biotechnology, October 2005, 1289-1293 and Benham,Nature Biotechnology, October 2005, 1234-1235, describe an approach forblocking ion channels with a six-transmembrane domain structure, inwhich polyclonal antibodies raised in rabbits directed against aspecific extracellular loop, i.e. the third extracellular region (E3),are used. However, rabbit polyclonal antibodies are not suited fortherapeutic use in human beings. It is thus an object of the presentinvention to provide therapeutic compounds that can be used in theprevention, treatment or diagnosis of diseases and disorders that areassociated with ion channels and/or with the biological and/orphysiological activity of ion channels. As used herein, “prevention” and“treatment” of a disease or disorder generally include any prophylacticor therapeutic effect that benefits a subject suffering or at risk ofthe disease or disorder, and for example also includes alleviating orpreventing one or more symptoms of the disease and preventing or slowingdown the onset and/or the (further) progression of the disease

In particular, it is an object of the present invention to provide suchtherapeutic compounds that are capable of modulating ion channels. By“modulating ion channels” is generally meant herein that the compound,upon coming into contact or otherwise suitably interacting with the ionchannel (i.e. under the conditions of a suitable in vitro, cellular orin vivo assay and/or in a suitable animal model; and in particular underphysiological conditions, i.e. upon suitable administration to asubject), provides an agonistic or antagonistic effect with respect tothe ion channel and/or with respect to the biological and/orphysiological functions associated with said ion channel.

It is another objective of the present invention to provide therapeuticcompounds that can be used in the prevention, treatment and/or diagnosisof diseases and disorders that can be treated, prevented and/ordiagnosed, respectively, by the use of the therapeutic compoundsdescribed herein in prophylaxis or therapy (i.e. by administering one ormore of the compounds to a subject in need of such treatment, as furtherdescribed herein) or for diagnostic purposes (also as further describedherein). In particular, it is an objective of the present invention toprovide therapeutic compounds that can be used in the prevention,treatment and/or diagnosis of diseases and disorders that can betreated, prevented and/or diagnosed, respectively, by modulating (asdefined herein) at least one ion channel. It is a further objective toprovide such compounds, which do not have the disadvantages that areassociated with the use of polyclonal antibodies, with the use ofantibodies that have been raised in rabbits, and with the use ofconventional four-chain antibodies. It is also an objective of thepresent invention to provide methods that can be used to easily generatesuch compounds.

One specific, but non-limiting object of the invention is to provideproteins and/or polypeptides directed against ion channels, and toprovide immunoglobulin sequences for use in such proteins orpolypeptides, that have improved therapeutic and/or pharmacologicalproperties and/or other advantageous properties (such as, for example,improved ease of preparation and/or reduced costs of goods), compared tothe conventional polyclonal rabbit antibodies described by Xu et al orfragments thereof. These improved and advantageous properties willbecome clear from the further description herein. The above objectivesare generally achieved by (the use of) the immunoglobulin sequences andcompositions described herein.

The immunoglobulin sequences, polypeptides and compositions of thepresent invention can generally be used to modulate the opening and/orclosing (or enhancing the opening and/or closing) of ion channels and/orto modulate the flow of ions through ion channels (i.e. to increase orto decrease such flow, or to partially or fully block such flow; in anirreversible manner but preferably in a reversible manner). As such, thepolypeptides and compositions of the present invention can generally beused to modulate the biological functions, pathways, responses, effects,mechanisms and actions in which ion channels and/or the flow of ionsthrough ion channels are involved. In particular, the polypeptides andcompositions of the invention may be used to reduce or inhibit (i.e.fully or partially, and in an irreversible manner but preferably in areversible manner) the flow of ions through ion channels. As such, theimmunoglobulin sequences, polypeptides and compositions of the presentinvention can generally act as (full or partial) blockers and/or as(full an partial) openers of ion channels (again in a an irreversiblemanner but preferably in a reversible manner) and/or as agonists or asantagonists of ion channels and/or of the biological functions,pathways, responses, effects, mechanisms and actions in which ionchannels and/or the flow of ions through ion channels are involved. Assuch, the polypeptides and compositions of the present invention can beused for the prevention and treatment (as defined herein) of diseasesand disorders associated with ion channels. Generally, “diseases anddisorders associated with ion channels” can be defined as diseases anddisorders that can be prevented and/or treated, respectively, bysuitably administering to a subject in need thereof (i.e. having thedisease or disorder or at least one symptom thereof and/or at risk ofattracting or developing the disease or disorder) either a polypeptideor composition of the invention (and in particular, of apharmaceutically active amount thereof) and/or a known active principleactive against ion channels. Examples of such diseases and disordersassociated with ion channels will be clear to the skilled person basedon the disclosure herein, and for example include the diseases anddisorders mentioned in the prior art cited herein, depending on the ionchannel(s) to which the immunoglobulin sequence or compound/polypeptideof the invention is directed. Thus, without being limited thereto, theimmunoglobulin sequences and polypeptides of the invention can forexample be used to prevent and/or to treat all diseases and disordersthat are currently being prevented or treated with active principlesthat can modulate ion channels, such as those mentioned in the prior artcited above. It is also envisaged that the polypeptides of the inventioncan be used to prevent and/or to treat all diseases and disorders forwhich treatment with such active principles is currently beingdeveloped, has been proposed, or will be proposed or developed infuture. In addition, it is envisaged that, because of their favourableproperties as further described herein, the polypeptides of the presentinvention may be used for the prevention and treatment of other diseasesand disorders than those for which these known active principles arebeing used or will be proposed or developed; and/or that thepolypeptides of the present invention may provide new methods andregimens for treating the diseases and disorders described herein.

Other applications and uses of the immunoglobulin sequences andpolypeptides of the invention will become clear to the skilled personfrom the further disclosure herein. In general, the invention providesimmunoglobulin sequences that are directed against (as defined herein)and/or can specifically bind (as defined herein) to ion channels; aswell as compounds and constructs, and in particular proteins andpolypeptides, that comprise at least one such immunoglobulin sequence.More in particular, the invention provides immunoglobulin sequences canbind to ion channels with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off) rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein;as well as compounds and constructs, and in particular proteins andpolypeptides, that comprise at least one such immunoglobulin sequence.

In particular, immunoglobulin sequences and polypeptides of theinvention are preferably such that they:

-   -   bind to ion channels with a dissociation constant (K_(D)) of        10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to ion channels with a k_(on)-rate of between 10² M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as        between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to ion channels with a k_(off) rate between 1s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent immunoglobulin sequence of the invention (or apolypeptide that contains only one immunoglobulin sequence of theinvention) is preferably such that it will bind to ion channels with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM. Some preferred IC50 valuesfor binding of the immunoglobulin sequences or polypeptides of theinvention to ion channels will become clear from the further descriptionand examples herein. For binding to ion channels, an immunoglobulinsequence of the invention will usually contain within its immunoglobulinsequence one or more amino acid residues or one or more stretches ofamino acid residues (i.e. with each “stretch” comprising two or aminoacid residues that are adjacent to each other or in dose proximity toeach other, i.e. in the primary or tertiary structure of theimmunoglobulin sequence) via which the immunoglobulin sequence of theinvention can bind to on channels, which amino acid residues orstretches of amino acid residues thus form the “site” for binding to ionchannels (also referred to herein as the “antigen binding site”).

The immunoglobulin sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more immunoglobulin sequences of theinvention and which may optionally further comprise one or more furtherimmunoglobulin sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or moreimmunoglobulin sequences of the invention may be used as a binding unitin such a protein or polypeptide, which may optionally contain one ormore further immunoglobulin sequences that can serve as a binding unit(i.e. against one or more other targets than ion channels), so as toprovide a monovalent, multivalent or multispecific polypeptide of theinvention, respectively, all as described herein. Such a protein orpolypeptide may also be in essentially isolated form (as definedherein).

The immunoglobulin sequences and polypeptides of the invention as suchpreferably essentially consist of a single amino acid chain that is notlinked via disulphide bridges to any other immunoglobulin sequence orchain (but that may or may not contain one or more intramoleculardisulphide bridges. For example, it is known that Nanobodies—asdescribed herein—may sometimes contain a disulphide bridge between CDR3and CDR1 or FR2). However, it should be noted that one or moreimmunoglobulin sequences of the invention may be linked to each otherand/or to other immunoglobulin sequences (e.g. via disulphide bridges)to provide peptide constructs that may also be useful in the invention(for example Fab′ fragments, F(ab′)₂ fragments, ScFv constructs,“diabodies” and other multispecific constructs. Reference is for examplemade to the review by Holliger and Hudson, Nat Biotechnol. 2005September; 23(9):1126-36)). Generally, when an immunoglobulin sequenceof the invention (or a compound, construct or polypeptide comprising thesame) is intended for administration to a subject (for example fortherapeutic and/or diagnostic purposes as described herein), it ispreferably either an immunoglobulin sequence that does not occurnaturally in said subject; or, when it does occur naturally in saidsubject, in essentially isolated form (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the immunoglobulin sequences of the invention (as well as compounds,constructs and polypeptides comprising the same) are preferably directedagainst human ion channels; whereas for veterinary purposes, theimmunoglobulin sequences and polypeptides of the invention arepreferably directed against ion channels from the species to be treated,or at least cross-reactive with ion channels from the species to betreated.

Furthermore, an immunoglobulin sequence of the invention may optionally,and in addition to the at least one binding site for binding against ionchannels, contain one or more further binding sites for binding againstother antigens, proteins or targets.

The efficacy of the immunoglobulin sequences and polypeptides of theinvention, and of compositions comprising the same, can be tested usingany suitable in vitro assay, cell-based assay, in vivo assay and/oranimal model known per se, or any combination thereof, depending on thespecific disease or disorder involved. Suitable assays and animal modelswill be clear to the skilled person, and for example include the assaysand animal models referred to in the reviews and prior art cited herein.

Also, according to the invention, immunoglobulin sequences andpolypeptides that are directed against ion channels from a first speciesof warm-blooded animal may or may not show cross-reactivity with ionchannels from one or more other species of warm-blooded animal. Forexample, immunoglobulin sequences and polypeptides directed againsthuman ion channels may or may not show cross reactivity with ionchannels from one or more other species of primates (such as, withoutlimitation, mouse, monkeys from the genus Macaca (such as, and inparticular, cynomolgus monkeys (Macaca fascicularis) and/or rhesusmonkeys (Macaca mulatta)) and baboon (Papio ursinus)) and/or with ionchannels from one or more species of animals that are often used inanimal models for diseases (for example mouse, rat, rabbit, pig or dog),and in particular in animal models for diseases and disorders associatedwith ion channels (such as the species and animal models mentionedherein). In this respect, it will be clear to the skilled person thatsuch cross-reactivity, when present, may have advantages from a drugdevelopment point of view, since it allows the immunoglobulin sequencesand polypeptides against human ion channels to be tested in such diseasemodels.

More generally, immunoglobulin sequences and polypeptides of theinvention that are cross-reactive with ion channels from multiplespecies of mammal will usually be advantageous for use in veterinaryapplications, since it will allow the same immunoglobulin sequence orpolypeptide to be used across multiple species. Thus, it is alsoencompassed within the scope of the invention that immunoglobulinsequences and polypeptides directed against ion channels from onespecies of animal (such as immunoglobulin sequences and polypeptidesagainst human ion channels) can be used in the treatment of anotherspecies of animal, as long as the use of the immunoglobulin sequencesand/or polypeptides provide the desired effects in the species to betreated.

The immunoglobulin sequences of the invention (as well as polypeptidescomprising the same) are preferably such that they are capable ofmodulating (as defined herein) an ion channel. In particular, theimmunoglobulin sequences of the invention (as well as polypeptidescomprising the same) are preferably such that they are capable of (fullyor partially) blocking an ion channel. By “partially or fully blocking”,respectively, of an ion channel by a compound is meant that themodulation of the ion channel by the immunoglobulin sequence orpolypeptide results in a reduced flow of ions through the channel (i.e.compared to the flow of ions through the ion channel when there is nointeraction between the immunoglobulin sequence/polypeptide and the ionchannel, as determined by a suitable assay, such as those describedherein) or essentially no flow of ions through the channel,respectively, irrespective of the specific mechanism via which the ionchannel is blocked by the immunoglobulin sequence or polypeptide. Forexample, and without limitation, the immunoglobulin sequence orpolypeptide may partially or fully block the ion channel directly bybinding to an epitope of the ion channel (or of at least one subunitthereof) such that the flow of ions through the channel is fully orpartially blocked (e.g. by steric hindrance or steric interactions); orindirectly, for example via interaction with one or more extracellularparts, regions, domain or loops of the ion channel or by affecting the(possible) confirmation(s) of the ion channel, or by limiting the extentand/or rate by which the ion channel can undergo conformational changes.

An immunoglobulin sequence or polypeptide of the invention that is ablocker of an ion channel is preferably such that it reduces the flow ofions through the channel by at least 1%, preferably at least 5%, such asat least 10%, fore example 25% or more or even 50% or more and up to 75%or even more than 90% or more, compared to the flow of ions through theion channel when there is no interaction between the immunoglobulinsequence/polypeptide and the ion channel, as determined by a suitableassay, such as those described herein.

An immunoglobulin sequence or polypeptide of the invention that is aopener of an ion channel is preferably such that it increases the flowof ions through the channel by at least 1%, preferably at least 5%, suchas at least 10%, fore example 25% or more or even 50% or more and up to75% or even more than 90% or more, compared to the flow of ions throughthe ion channel when there is no interaction between the immunoglobulinsequence/polypeptide and the ion channel, as determined by a suitableassay, such as those described herein.

An immunoglobulin sequence or polypeptide of the invention that is anagonists of an ion channel and/or of the biological function(s) orresponse(s) associated therewith is preferably such that it increasesthe desired biological function or response by at least 1%, preferablyat least 5%, such as at least 10%, fore example 25% or more or even 50%or more and up to 75% or even more than 90% or more, compared to thebiological function or response when there is no interaction between theimmunoglobulin sequence/polypeptide and the on channel, as determined bya suitable assay, such as those described herein.

An immunoglobulin sequence or polypeptide of the invention that is anantagonists of an ion channel and/or of the biological function(s) orresponse(s) associated therewith is preferably such that it decreasesthe desired biological function or response by at least 1%, preferablyat least 5%, such as at least 10%, fore example 25% or more or even 50%or more and up to 75% or even more than 90% or more, compared to thebiological function or response when there is no interaction between theimmunoglobulin sequence/polypeptide and the ion channel, as determinedby a suitable assay, such as those described herein.

Immunoglobulin sequences or polypeptides of the invention that areblockers or openers of ion channels (or enhance blockers or openers)and/or that are agonists or antagonists of ion channels or thebiological function(s) or response(s) associated therewith may providetheir desired activity in an irreversible manner, but preferably do soin a reversible manner.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of ion channelsagainst which the immunoglobulin sequences and polypeptides of theinvention are directed.

The immunoglobulin sequences and polypeptides of the invention may bedirected against any desired ion channel, and may in particular bedirected against an ion channel that has at least one extracellulardomain. More in particular, but without limitation, the immunoglobulinsequences and polypeptides of the invention may be directed against anydesired ion channel that has at least one transmembrane domain, more inparticular at least two transmembrane domains, such as at least fourtransmembrane domains (e.g., the 4-TM ion channels) or six or moretransmembrane domains (e.g. the 6-TM ion channels). Examples of such ionchannels will be clear to the skilled person based on the prior artcited herein. Reference is for example made to the reviews fromGoldstein et al., Yu et al., Clapham and Garbers, Hoffmann et al., Kuboet al., Wei et al., Shieh et al. (see for example FIG. 2 on page 559),Catterall et al, Gutman et al., Clapham et al., Catterall et al. citedabove. As further described herein, the immunoglobulin sequences andpolypeptides of the invention may for example be directed against theextracellular parts of such transmembrane domains and in particularagainst the extracellular loops/repeats that connect such transmembranedomains (reference is for example again made to FIG. 2 on page 559 ofShieh et al. and to FIG. 1 on page 1290 of Xu et al, supra, whichschematically show such extracellular loops of 2-TM, 4-TM and 6-TM ionchannels).

In one preferred, but non-limiting aspect, the immunoglobulin sequencesand polypeptides of the invention are directed against 6-TM ion channels(such as, without limitation, the Kv-ion channels mentioned above (e.g.from Kv1.1 to Kv9.3, the KvLQT ion channels, Slo and IK_(Ca)10), and inparticular against one of the extracellular loops/repeats that connectthe transmembrane domains of such 6-TM ion channels, such as the E1, E2,and in particular E3 loop, or the region between 85 and 86 of repeats 2,3 and 4.

However, according to a specific aspect of the invention, animmunoglobulin sequence or polypeptide of the invention may be directedagainst (as defined herein) an ion channel that is expressed on thesurface of a cell and/or against at least one extracellular region,domain, loop or other extracellular epitope of an ion channel.

In particular, according to this aspect, an immunoglobulin sequence orpolypeptide of the invention is directed against (as defined herein) atleast one extracellular region, domain, loop or other extracellularepitope of an ion channel; and is preferably further such that saidimmunoglobulin sequence or polypeptide of the invention is capable ofmodulating (as defined herein) said ion channel. More in particular,according to this aspect, an immunoglobulin sequence or polypeptide ofthe invention is directed against (as defined herein) at least oneextracellular region, domain, loop or other extracellular epitope of anion channel; and is preferably further such that said immunoglobulinsequence or polypeptide of the invention is capable of (fully orpartially) blocking said ion channel.

According to this aspect of the invention, the immunoglobulin sequenceor polypeptide of the invention may be directed against any suitableextracellular part, region, domain, loop or other extracellular epitope,but is preferably directed against one of the extracellular parts of thetransmembrane domains or more preferably against one of theextracellular loops that link the transmembrane domains. More inparticular, when the ion channel is a 6-TM channel, the protein orpolypeptide (and in particular at least one of the immunoglobulinsequences present therein) may be directed against the extracellular E3loop that linking the transmembrane domains (and/or may have been raisedagainst the extracellular E3 loop and/or against synthetic orsemi-synthetic peptides that are derived from or based on the sequenceof the extracellular E3 loop).

Other suitable extracellular parts, regions, domains, loops or epitopesmay be derived by Kyte-Doolittle analysis of the immunoglobulin sequenceof the pertinent ion channel; by aligning ion channels belonging to thesame (sub)families and identifying the various transmembrane domains andextracellular parts, regions, domain or loops (including the E3 loop);by TMAP-analysis; or by any suitable combination thereof. The inventionalso relates to immunoglobulin sequences and (as further defined herein)that are directed against such extracellular parts, regions, domains,loops or epitopes (and/or that have been raised against parts orfragments of the immunoglobulin sequence that comprise suchextracellular parts, regions, domains, loops or epitopes and/or againstsynthetic or semi-synthetic peptides that are derived from or based onsuch extracellular parts, regions, domains, loops or epitopes).

In particular, immunoglobulin sequences and polypeptides of theinvention are preferably such that they:

-   -   bind to an extracellular part, region, domain, loop or other        extracellular epitope of an ion channel (as described herein)        with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²        moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or        less and more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an        association constant (K_(A)) of 10⁵ to 10¹² liter/moles or more,        and preferably 10⁷ to 10¹² liter/moles or more and more        preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to an extracellular part, region, domain, loop or other        extracellular epitope of an ion channel (as described herein)        with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,        preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably        between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵M⁻¹s⁻¹ and        10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to an extracellular part, region, domain, loop or other        extracellular epitope of an ion channel (as described herein)        with a k_(off) rate between 1s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁸ s⁻¹        (providing a near irreversible complex with a t_(1/2) of        multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more        preferably between 10⁻³ s⁻¹ and 10⁻⁸ s⁻¹, such as between 10⁻⁴        s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent immunoglobulin sequence of the invention (or apolypeptide that contains only one immunoglobulin sequence of theinvention) is preferably such that it will bind to bind to anextracellular part, region, domain, loop or other extracellular epitopeof an ion channel (as described herein) with an affinity less than 500nM, preferably less than 200 nM, more preferably less than 10 nM, suchas less than 500 pM.

Some preferred 1050 values for binding of the immunoglobulin sequencesor polypeptides of the invention to bind to an extracellular part,region, domain, loop or other extracellular epitope of an ion channel(as described herein) will become clear from the further description andexamples herein.

Also, according to this aspect, any multivalent or multispecific (asdefined herein) polypeptides of the invention may also be suitablydirected against two or more different extracellular parts, regions,domains, loops or other extracellular epitopes on the same antigen, forexample against two different extracellular loops, against two differentextracellular parts of the transmembrane domains or against oneextracellular loops and one extracellular loop. Such multivalent ormultispecific polypeptides of the invention may also have (or beengineered and/or selected for) increased avidity and/or improvedselectivity for the desired ion channel, and/or for any other desiredproperty or combination of desired properties that may be obtained bythe use of such multivalent or multispecific polypeptides.

An immunoglobulin sequence or polypeptide of the invention may also besaid to be “directed against” (as further defined herein) a peptideantigen when it is directed against said peptide antigen per se, forexample in a standard assay for determining the binding (i.e. thespecificity, affinity, K_(D), K_(A), k_(on) or k_(off) of such binding;all as described herein) of the immunoglobulin sequence or polypeptideof the invention against the peptide antigen, using the peptide antigenof the invention as such (instead of, for example, the peptide antigenas part of a larger protein or polypeptide, for example as part of anion channel present on the surface of a cell). Techniques fordetermining the binding of immunoglobulin sequences or polypeptides tosmall peptides will be clear to the skilled person, and for exampleinclude the techniques described herein.

An immunoglobulin sequence or polypeptide of the invention may also besaid to be “directed against” (as further defined herein) a peptideantigen when it has been screened against, selected using and/or raisedagainst (i.e. by suitably immunizing a mammal, as further describedherein) said peptide antigen. Techniques for raising immunoglobulinsequences and polypeptides of the invention against a peptide antigen ofthe invention, and for screening or selecting immunoglobulin sequencesand polypeptides of the invention for binding against a peptide antigenof the invention, will be clear to the skilled person, for example basedon the further disclosure herein.

Generally, it is expected that immunoglobulin sequences an polypeptidesof the invention that are directed against a peptide antigen of theinvention per se, and/or that have been screened against, selected usingand/or raised against a peptide antigen of the invention, will also beable to bind (and in particular, to specifically bind, as definedherein) to a peptide antigen of the invention that forms part of an ionchannel (or at least one subunit thereof) that is present on the surfaceof a cell. Thus, the peptide antigens of the invention may findparticular use in methods for generating immunoglobulin sequences andpolypeptides of the invention (as defined herein); and such methods anduses form further aspects of the invention.

For example, such a method may comprise one of the following steps or asuitable combination of both of the following steps:

-   a) a step of suitably immunizing a Camelid with a suitable antigen    that comprises the desired extracellular part, region, domain, loop    or other extracellular epitope(s), such that an immune response    against the desired extracellular part, region, domain, loop or    other extracellular epitope(s) is raised. The antigen may be any    suitable antigen that is capable of raising an immune response    against the desired extracellular part, region, domain, loop or    other extracellular epitope(s); such as, for example and without    limitation, whole cells that have the desired extracellular part,    region, domain, loop or other extracellular epitope(s) on their    surface, cell wall fragments thereof or any other suitable    preparation derived from such cells, vesicles that have the desired    extracellular part, region, domain, loop or other extracellular    epitope(s) on their surface, a subunit or fragment of a subunit of    an ion channel that comprises the desired extracellular part,    region, domain, loop or other extracellular epitope(s), or a    synthetic or semi-synthetic peptide that comprises and/or is based    on (the immunoglobulin sequence of) the desired extracellular part,    region, domain, loop or other extracellular epitope(s);    and/or-   b) a step of screening for affinity and/or binding for the desired    extracellular part, region, domain, loop or other extracellular    epitope(s). This may for example be performed by screening a set,    collection or library of cells that express heavy chain antibodies    on their surface (e.g. B-cells obtained from a suitably immunized    Camelid), by screening of a (naïve or immune) library of VHH    sequences or Nanobody sequences, or by screening of a (naïve or    immune) library of nucleic acid sequences that encode VHH sequences    or Nanobody sequences; which may all be performed in a manner known    per se, for which reference is made to the further disclosure and    prior art mentioned herein;    and which method may optionally further comprise one or more other    suitable steps known per se, such as, for example and without    limitation, a step of affinity maturation, a step of expressing the    desired immunoglobulin sequence, a step of screening for binding    and/or for activity against the desired antigen (in this case, the    ion channel), a step of determining the desired immunoglobulin    sequence or nucleotide sequence, a step of introducing one or more    humanizing substitutions (e.g. as further described herein), a step    of formatting in a suitable multivalent and/or multispecific format,    a step of screening for the desired biological and/or physiological    properties (i.e. using a suitable assay, such as those described    herein); and/or any suitable combination of one or more of such    steps, in any suitable order.

Such methods and the immunoglobulin sequences obtained via such methods,as well as proteins and polypeptides comprising or essentiallyconsisting of the same, form further aspects of this invention.

In a preferred embodiment, the immunoglobulin sequence or polypeptide ofthe invention is a “monoclonal” immunoglobulin sequence or polypeptide,by which is meant that at least each of the one or more immunoglobulinsequences directed against the ion channel that are present in saidprotein or polypeptide (and preferably all of the immunoglobulinsequences that are present in said protein or polypeptide) are“monoclonal” as commonly understood by the skilled person. In thisrespect, it should however be noted that, as further described herein,the present invention explicitly covers multivalent or multispecificproteins that comprise two or more immunoglobulin sequences (and inparticular monoclonal immunoglobulin sequences) that are directedagainst different parts, regions, domains, loops or epitopes of the sameion channel, and in particular against different extracellular parts,regions, domains, loops or epitopes of the same ion channel.

In another aspect, the invention relates to a protein or polypeptidethat comprises or essentially consist of at least one immunoglobulinsequence of the invention, or of at least one part, fragment, analog,variant or derivative of an immunoglobulin sequence of the invention,wherein said protein or polypeptide is capable of modulating (as definedherein) an ion channel. Preferably, said protein or polypeptide iscapable of fully or partially blocking (as defined herein) an ionchannel.

The protein or polypeptides described herein are preferably directedagainst an ion channel that is expressed on the surface of a cell and/oragainst at least one extracellular region, domain, loop or otherextracellular epitope of an ion channel, more preferably against atleast one extracellular loop of an ion channel. In one specific aspect,when the ion channel is an ion channel with six transmembrane domains(6-TM), the protein or polypeptide is preferably directed against theextracellular E3 loop. The proteins or polypeptides described herein mayalso be directed against a peptide antigen of the invention.

In one specific aspect, the invention relates to a protein orpolypeptide that comprises or essentially consist of at least oneimmunoglobulin sequence of the invention, or of at least one part,fragment, analog, variant or derivative of an immunoglobulin sequence ofthe invention, wherein at least one of the immunoglobulin sequences ofthe invention (or at least one of the parts, fragments, analogs,variants or derivatives of the immunoglobulin sequence of the invention)present in protein or polypeptide is directed against an ion channel.Preferably, at least one of the immunoglobulin sequences of theinvention (or at least one of the parts, fragments, analogs, variants orderivatives of the immunoglobulin sequence of the invention) present inprotein or polypeptide is capable of modulating an ion channel, and morepreferably of fully or partially blocking an ion channel. Also,preferably, at least one of the immunoglobulin sequences of theinvention (or at least one of the parts, fragments, analogs, variants orderivatives of the immunoglobulin sequence of the invention) present inprotein or polypeptide is directed against at least one extracellularregion, domain, loop or other extracellular epitope of an ion channel,and in particular against one extracellular loop of an ion channel. Inone specific aspect, when the ion channel is an ion channel with sixtransmembrane domains (6-TM), at least one of the immunoglobulinsequences of the invention present in the protein or polypeptide ispreferably directed against the extracellular E3 loop.

The proteins or polypeptides described herein may comprise oressentially consist of a single immunoglobulin sequence of the invention(or part, fragment, analog, variant or derivative of an immunoglobulinsequence of the invention), or alternatively of at least two (such astwo, three, four or more) immunoglobulin sequences of the invention (orparts, fragments, analogs, variants or derivatives of an immunoglobulinsequence of the invention), which are optionally suitably linked via oneor more suitable linkers (as described herein). Suitable examples ofsuch linkers will be clear to the person skilled in the art, for exampleon the basis of the further disclosure herein.

In one aspect of the invention, when a protein or polypeptide comprisesat least two immunoglobulin sequences of the invention, at least two ofthe immunoglobulin sequences of the invention (or parts, fragments,analogs, variants or derivatives of an immunoglobulin sequence of theinvention) present in the protein or polypeptide are directed againstthe same ion channel. For example, at least two of the immunoglobulinsequences of the invention (or parts, fragments, analogs, variants orderivatives of an immunoglobulin sequence of the invention) present inthe protein or polypeptide may directed against different extracellularregions, domains, loops or other extracellular epitopes of the same ionchannel. However, preferably at least one (or at least two) of theimmunoglobulin sequences of the invention (or parts, fragments, analogs,variants or derivatives of an immunoglobulin sequence of the invention)present in the protein or polypeptide is directed against at least oneextracellular loop of the ion channel.

When a protein or polypeptide of the invention comprises or essentiallyconsists of at least two immunoglobulin sequences of the invention (orparts, fragments, analogs, variants or derivatives of an immunoglobulinsequence of the invention), it may comprise at least two differentimmunoglobulin sequences of the invention (or parts, fragments, analogs,variants or derivatives of an immunoglobulin sequence of the invention),and/or comprise at least two identical immunoglobulin sequences of theinvention (or parts, fragments, analogs, variants or derivatives of animmunoglobulin sequence of the invention).

In one specific aspect, when a protein or polypeptide of the inventioncomprises or essentially consists of at least two immunoglobulinsequences of the invention (or parts, fragments, analogs, variants orderivatives of an immunoglobulin sequence of the invention), it maycomprise at least one immunoglobulin sequence of the invention (or part,fragment, analog, variant or derivative of an immunoglobulin sequence ofthe invention) that is directed against a protein, polypeptide or otherantigen different from an ion channel.

In another aspect, the invention relates to an immunoglobulin sequenceof the invention, or part, fragment, analog, variant or derivative of animmunoglobulin sequence of the invention that is capable of modulatingan ion channel. Preferably, said immunoglobulin sequence of theinvention is capable of fully or partially blocking (as defined herein)an ion channel.

The immunoglobulin sequences of the invention described herein arepreferably directed against at least one extracellular region, domain,loop or other extracellular epitope of an ion channel, more preferablyagainst at least one extracellular loop of an ion channel. In onespecific aspect, when the ion channel is an ion channel with sixtransmembrane domains (6-TM), the immunoglobulin sequence of theinvention is preferably directed against the extracellular E3 loop.

It is also within the scope of the invention that, where applicable, animmunoglobulin sequence of the invention can bind to two or moreantigenic determinants, epitopes, parts, domains, subunits orconfirmations of ion channels. In such a case, the antigenicdeterminants, epitopes, parts, domains or subunits of ion channels towhich the immunoglobulin sequences and/or polypeptides of the inventionbind may be essentially the same (for example, if ion channels containsrepeated structural motifs or occurs in a multimeric form) or may bedifferent (and in the latter case, the immunoglobulin sequences andpolypeptides of the invention may bind to such different antigenicdeterminants, epitopes, parts, domains, subunits of ion channels with anaffinity and/or specificity which may be the same or different). Also,for example, when ion channels exists in an activated conformation andin an inactive conformation, the immunoglobulin sequences andpolypeptides of the invention may bind to either one of theseconfirmation, or may bind to both these confirmations (i.e. with anaffinity and/or specificity which may be the same or different). Also,for example, the immunoglobulin sequences and polypeptides of theinvention may bind to a conformation of ion channels in which it isbound to a pertinent ligand, may bind to a conformation of ion channelsin which it not bound to a pertinent ligand, or may bind to both suchconformations (again with an affinity and/or specificity which may bethe same or different).

It is also expected that the immunoglobulin sequences and polypeptidesof the invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments ofion channels; or at least to those analogs, variants, mutants, alleles,parts and fragments of ion channels that contain one or more antigenicdeterminants or epitopes that are essentially the same as the antigenicdeterminant(s) or epitope(s) to which the immunoglobulin sequences andpolypeptides of the invention bind in ion channels (e.g. in wild-typeion channels). Again, in such a case, the immunoglobulin sequences andpolypeptides of the invention may bind to such analogs, variants,mutants, alleles, parts and fragments with an affinity and/orspecificity that are the same as, or that are different from (i.e.higher than or lower than), the affinity and specificity with which theimmunoglobulin sequences of the invention bind to (wild-type) ionchannels. It is also included within the scope of the invention that theimmunoglobulin sequences and polypeptides of the invention bind to someanalogs, variants, mutants, alleles, parts and fragments of ionchannels, but not to others.

When ion channels exists in a monomeric form and in one or moremultimeric forms, it is within the scope of the invention that theimmunoglobulin sequences and polypeptides of the invention only bind toion channels in monomeric form, only bind to ion channels in multimericform, or bind to both the monomeric and the multimeric form. Again, insuch a case, the immunoglobulin sequences and polypeptides of theinvention may bind to the monomeric form with an affinity and/orspecificity that are the same as, or that are different from (i.e.higher than or lower than), the affinity and specificity with which theimmunoglobulin sequences of the invention bind to the multimeric form.

Also, when ion channels can associate with other proteins orpolypeptides to form protein complexes (e.g. with multiple subunits), itis within the scope of the invention that the immunoglobulin sequencesand polypeptides of the invention bind to ion channels in itsnon-associated state, bind to ion channels in its associated state, orbind to both. In all these cases, the immunoglobulin sequences andpolypeptides of the invention may bind to such multimers or associatedprotein complexes with an affinity and/or specificity that may be thesame as or different from (i.e. higher than or lower than) the affinityand/or specificity with which the immunoglobulin sequences andpolypeptides of the invention bind to ion channels in its monomeric andnon-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more immunoglobulin sequences directed against ionchannels may bind with higher avidity to ion channels than thecorresponding monomeric immunoglobulin sequence(s). For example, andwithout limitation, proteins or polypeptides that contain two or moreimmunoglobulin sequences directed against different epitopes of ionchannels may (and usually will) bind with higher avidity than each ofthe different monomers, and proteins or polypeptides that contain two ormore immunoglobulin sequences directed against ion channels may (andusually will) bind also with higher avidity to a multimer of ionchannels.

Generally, immunoglobulin sequences and polypeptides of the inventionwill at least bind to those forms of ion channels (including monomeric,multimeric and associated forms) that are the most relevant from abiological and/or therapeutic point of view, as will be clear to theskilled person. It is also within the scope of the invention to useparts, fragments, analogs, mutants, variants, alleles and/or derivativesof the immunoglobulin sequences and polypeptides of the invention,and/or to use proteins or polypeptides comprising or essentiallyconsisting of one or more of such parts, fragments, analogs, mutants,variants, alleles and/or derivatives, as long as these are suitable forthe uses envisaged herein. Such parts, fragments, analogs, mutants,variants, alleles and/or derivatives will usually contain (at least partof) a functional antigen-binding site for binding against ion channels;and more preferably will be capable of specific binding to ion channels,and even more preferably capable of binding to ion channels with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Some non-limiting examples of suchparts, fragments, analogs, mutants, variants, alleles, derivatives,proteins and/or polypeptides will become clear from the furtherdescription herein. Additional fragments or polypeptides of theinvention may also be provided by suitably combining (i.e. by linking orgenetic fusion) one or more (smaller) parts or fragments as describedherein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the immunoglobulin sequence from which they havebeen derived. For example, an immunoglobulin sequence of the inventionmay be linked (chemically or otherwise) to one or more groups ormoieties that extend the half-life (such as PEG), so as to provide aderivative of an immunoglobulin sequence of the invention with increasedhalf-life.

In one specific, but non-limiting aspect, the immunoglobulin sequence ofthe invention may be an immunoglobulin sequence that comprises animmunoglobulin fold or may be an immunoglobulin sequence that, undersuitable conditions (such as physiological conditions) is capable offorming an immunoglobulin fold (i.e. by folding). Reference is interalia made to the review by Halaby et al., J. (1999) Protein Eng. 12,563-71. Preferably, when properly folded so as to form an immunoglobulinfold, such an immunoglobulin sequence is capable of specific binding (asdefined herein) to ion channels; and more preferably capable of bindingto ion channels with an affinity (suitably measured and/or expressed asa K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent),a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value,as further described herein) that is as defined herein. Also, parts,fragments, analogs, mutants, variants, alleles and/or derivatives ofsuch immunoglobulin sequences are preferably such that they comprise animmunoglobulin fold or are capable for forming, under suitableconditions, an immunoglobulin fold.

In particular, but without limitation, the immunoglobulin sequences ofthe invention may be immunoglobulin sequences that essentially consistof 4 framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an immunoglobulin sequence (which will then usuallycontain at least some of the amino acid residues that form at least oneof the CDR's, as further described herein).

In such an immunoglobulin sequence of the invention, the frameworksequences may be any suitable framework sequences, and examples ofsuitable framework sequences will be clear to the skilled person, forexample on the basis the standard handbooks and the further disclosureand prior art mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the immunoglobulinsequence of the invention is a domain antibody (or an immunoglobulinsequence that is suitable for use as a domain antibody); is a singledomain antibody (or an immunoglobulin sequence that is suitable for useas a single domain antibody); is a “dAb” (or an immunoglobulin sequencethat is suitable for use as a dAb); or is a Nanobody™ (including but notlimited to V_(HH) sequence). Again, suitable framework sequences will beclear to the skilled person, for example on the basis the standardhandbooks and the further disclosure and prior art mentioned herein.

In particular, the framework sequences present in the immunoglobulinsequences of the invention may contain one or more of Hallmark residues(as defined herein), such that the immunoglobulin sequence of theinvention is a Nanobody™. Some preferred, but non-limiting examples of(suitable combinations of) such framework sequences will become clearfrom the further disclosure herein.

Again, as generally described herein for the immunoglobulin sequences ofthe invention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence).

Such a fragment may also contain a disulphide bridge, and in particulara disulphide bridge that links the two framework regions that precedeand follow the CDR sequence, respectively (for the purpose of formingsuch a disulphide bridge, cysteine residues that naturally occur in saidframework regions may be used, or alternatively cysteine residues may besynthetically added to or introduced into said framework regions). For afurther description of these “Expedite fragments”, reference is againmade to WO 03/050531, as well as to WO 2009/127691.

The immunoglobulin sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the immunoglobulin sequence of the invention is a heavychain variable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the immunoglobulin sequence of the invention (or of thenucleotide sequence of the invention used to express it), nor as to theway that the immunoglobulin sequence or nucleotide sequence of theinvention is (or has been) generated or obtained. Thus, theimmunoglobulin sequences of the invention may be naturally occurringimmunoglobulin sequences (from any suitable species) or synthetic orsemi-synthetic immunoglobulin sequences. In a specific but non-limitingaspect of the invention, the immunoglobulin sequence is a naturallyoccurring immunoglobulin sequence (from any suitable species) or asynthetic or semi-synthetic immunoglobulin sequence, including but notlimited to “humanized” (as defined herein) immunoglobulin sequences(such as partially or fully humanized mouse or rabbit immunoglobulinsequences, and in particular partially or fully humanized V_(HH)sequences or Nanobodies), “camelized” (as defined herein) immunoglobulinsequences, as well as immunoglobulin sequences that have been obtainedby techniques such as affinity maturation (for example, starting fromsynthetic, random or naturally occurring immunoglobulin sequences), CDRgrafting, veneering, combining fragments derived from differentimmunoglobulin sequences, PCR assembly using overlapping primers, andsimilar techniques for engineering immunoglobulin sequences well knownto the skilled person; or any suitable combination of any of theforegoing. Reference is for example made to the standard handbooks, aswell as to the further description and prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

In particular, the immunoglobulin sequence of the invention may be aNanobody™ (as defined herein) or a suitable fragment thereof. [Note:Nanobody™, Nanobodies™ and Nanoclone™ are trademarks of Ablynx N.V.]Such Nanobodies directed against ion channels will also be referred toherein as “Nanobodies of the invention”. For a general description ofNanobodies, reference is made to the further description below, as wellas to the prior art cited herein. In this respect, it should however benoted that this description and the prior art mainly describedNanobodies of the so-called “V_(H)3 class” (i.e. Nanobodies with a highdegree of sequence homology to human germline sequences of the V_(H)3class such as DP-47, DP-51 or DP-29), which Nanobodies form a preferredaspect of this invention. It should however be noted that the inventionin its broadest sense generally covers any type of Nanobody directedagainst ion channels, and for example also covers the Nanobodiesbelonging to the so-called “V_(H)4 class” (i.e. Nanobodies with a highdegree of sequence homology to human germline sequences of the V_(H)4class such as DP-78), as for example described in the U.S. provisionalapplication 60/792,279 by Ablynx N.V. entitled “DP-78-like Nanobodies”filed on Apr. 14, 2006.

Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein). Thus,generally, a Nanobody can be defined as an immunoglobulin sequence withthe (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which one or more of the Hallmark residues are as further        defined herein.

In particular, a Nanobody can be an immunoglobulin sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which the framework sequences are as further defined herein.        In particular, a Nanobody can be an immunoglobulin sequence with        the (general) structure    -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which the framework sequences are as further defined herein.

More in particular, a Nanobody can be an immunoglobulin sequence withthe (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 below;    and in which:

-   ii) said immunoglobulin sequence has at least 80% amino acid    identity with at least one of the immunoglobulin sequences of SEQ ID    NO's: 1 to 22, in which for the purposes of determining the degree    of amino acid identity, the amino acid residues that form the CDR    sequences (indicated with X in the sequences of SEQ ID NO's: 1    to 22) are disregarded.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Thus, the invention also relates to such Nanobodies that can bind to (asdefined herein) and/or are directed against an ion channel, to suitablefragments thereof, as well as to polypeptides that comprise oressentially consist of one or more of such Nanobodies and/or suitablefragments.

SEQ ID NO's: 705 to 788, more preferably SEQ ID NO's 726 to 750, 753 to758, 762 to 764, 772 to 773, 775, or 778 to 780 (see Table A-1) give theimmunoglobulin sequences of a number of V_(HH) sequences that have beenraised against an ion channel.

TABLE A-1 Preferred VHH sequences or Nanobody sequences (also referredherein as a sequence with a particular name or SEQ ID NO: X,wherein X is a number referring to the relevant immunoglobulinsequence): SEQ ID NO: X, wherein P2X7 Name X = function FamilyIMMUNGLOBULIN SEQUENCE P2X7PMP2C4 705 binder 1EVQLVESGGDVVQAGGSLRLSCLASGFT FDDYAIGWFRQAPGKEREGISCISSTGNVFYADSVKGRFTISSDKEKNTLYLQMNSLK PEDTAVYHCAAGHFTVDSGKVLLRTDISS WGQGTQVTVSSP2X7PMP2A6 706 Binder 1 EVQLVESGGDVVQAGGSLRLSCLASGFTFDDYAIGWFRQAPGKEREGISCISSTGNV FYADSVKGRFTISSDKEKNTLYLQMNSLEPEDTAVYHCAAGHFTVDSGKVLLRTDISS WGQGTQVTVSS P2X7PMP4D5 707 Binder 1EMQLVESGGDVVQAGGSLRLSCLASGFT FDDYAIGWFRQAPGKEREGISCISSTGNVFYADSVKGRFTISSDKEKNTLYLQMNSLK PEDTAVYHCAAGHFTVDSGKVLLRTDISS WGQGTQVTVSSP2X7PMP20E8 708 Binder 1 EVQLVESGGDVVQAGGSLRLSCLASGFTFDDYAIGWFRQAPGKEREGISCISSTGNV FYADSVKGRFTISSDKEKNTLYLQMNSLKPGDTAVYHCAAGHFTVDSGKVLLRTDISS WGQGTQVTVSS P2X7PMP16H8 709 Binder 1EVQLVESGGDVVQAGGSLRLSCLASGFT FDDYAIGWFRQAPGKEREGISCISSTGNVFYADSVKGRLTISSDKEKNTLYLQMNSLK PEDTAVYHCAAGHFTVDSGKVLLRTDISS WGQGTQVTVSSP2X7PMP16B10 710 Binder 1 EVQLVESGGDVVQAGGSLRLSCLASGFTFDDYAIGWFRQAPGKEREGISCISSTGNV FYADSVKGRFTISSDKEKNTLYLQMDSLKPEDTAVYHCAAGHFTVDSGKVLLRTDISS WGQGTQVTVSS P2X7PMP4G6 711 binder 1EVQLVESGGDVVQAGGSLRLSCLASGFT FDDYAIGWFRQAPGKEREGISCISSTGNVFYADSVKGRFTISSDKEKNTLYLQMNSLK PEDTAVYHCAAGHFTVDSGKVLLRTDVS SWGQGTQVTVSSP2X7PMP1A8 712 Binder 2 EVQLVESGGGLVQTGGSLRLSCAASGFTLDDYAIAWFRQAPGKEREGVSILSSIGKT FYADSVKDRFSITADGAKTTVFLQMNSLKPGDTAIYYCVAGHFVYNDGAISLNTARGS GFWGQGAQVTVSS P2X7PMP1C9 713 Binder 2EVQLVESGGGLVQTGGSLRLSCAASGFT LDDYAIAWFRQAPGKEREGVSILSSIGKTFYADSVKDRFSITADGAKTTVFLQMNSLK PEDTAIYYCVAGHFVYNDGAISLNTARGSGFWGQGAQVTVSS P2X7PMP20B10 714 Binder 2 EVQLVESGGGLVQTGGSLRLSCAASGFTLDDYAIAWFRQAPGKEREGVSILSSIGKT FYADSVKDRFSITADGAKTTVFLQMNSLKPEDTAIYYCVAGHFVYNDGAISLNTARGS GFWGQGTQVTVSS P2X7PMP20C9 715 Binder 2EVQLVESGGGLVQTGGSLRLSCAASGFT LDDYAIAWFRQAPGKEREGVSILSSIGKTFYADSAKDRFSITADGAKTTVFLQMNSLK PEDTAIYYCVAGHFVYNDGAISLNTARGSGFWGQGTQVTVSS P2X7PMP5A1 716 Binder 3 EVQLVESGGGLVQAGGSLRLSCAASERTYSMGWFRQAPGKEREFVAGSGWDGIPT RYADSVKGRLTISRDNAKNTVSLQMSGLKPEDTAIYYCATGTSVYHYQYWGQGTQV TVSS P2X7PMP5B1 717 binder 3EVQLVESGGGLVQAGGSLRLSCAASERT YSMGWFRQAPGKEREFVAGSGWDGIPTRYADSVKGRFTISRDNAKNTVSLQMSGL KPEDTAIYYCATGTSVYHYQYRGQGTQV TVSSP2X7PMP11G1 718 Binder 3 EVQLVESGGGLVQAGGSLRLSCAASERTYSMGWFRRAPGKEREFVAGSGWDGIPT RYADSVKGRFTISRDNAKNTVSLQMSGLKPEDTAIYYCATGTSVYHYQYWGQGTQV TVSS P2X7PMP11A1 719 Binder 3EVQLVESGGGLVQAGGSLRLSCAASERT YSVGWFRQAPGKEREFVAGSGWDGTPTRYADSVKGRFTISRDNAKNTVSLQMSGL KPEDTAIYYCATGTSVYHYQYWGQGTQV TVSSP2X7PMP7E2 720 Binder 3 EVQLVESGGGLVQAGGSLRLSCAAPERTYSMGWFRQAPGKEREFVAGSGWDGIPT RYADSVKGRFTISRDNAKNTVSLQMSGLKPEDTAIYYCATGTSVYHYQYWGQGTQV TVSS P2X7PMP5F1 721 Binder 3EVQLVESGGGLVQAGGSLRLSCAASERT YSMGWFRQAPGKEREFVAGSGWDGIPTRYADSVKGRFTISRDNAKNTVSLQMSGL KPEDTAIYYCATGTSVYHYQYWGQGTQV TVSSP2X7PMP7F3 722 Binder 3 EVQLVESRGGLVQAGGSLRLSCAASERTYSMGWFRQAPGKEREFVAGSGWDGIPT RYADSVKSRFTISRDNAKNTVSLQMSGLKPEDTAIYYCATGTSVYHYQYWGQGTQVT VSS P2X7PMP13B2 723 binder 3EVQLVESGGGLVQAGGSLRLSCAASERT YSMGWFRQAPGKEREFVAGSGWDGIPTRYADSVKGRFTISRDNAKNAVSLQMSGL KPEDTAIYYCATGTSVYHYQYWGQGTQV TVSSP2X7PMP11D3 724 Binder 3 EVQLVESGGGLVQPGGSLRLSCAASERTYSMGWFRQAPGKEREFVAGSGWDGIPT RYADSVKGRFTISRDNAKNTVSLQMSGLKPEDTAIYYCATGTSVYHYQYWGQGTQV TVSS P2X7PMP7F1 725 Binder 3EVQLVESGGGLVQAGGSLRLSCAASERT YSMGWFRQAPGKEREFVAGSGWDGIPTRYADSVKGRFTISRDNAKSTVSLQMSGL KPEDTAIYYCATGTSVYHYQYWGQGTQV TVSSP2X7PMP4B4 726 Enhancer 4 EVQLVESGGGLVQAGGSLRLSCAASGRTVSDYGMGWFRQAPGKLREFVASINWSGI YTRYIDSVEGRFTISRDNTKNTLYLQMNNLKAEDTAVYYCAYFLGPNWYSNYGRPSS YDFYGQGTQVTVSS P2X7PMP19A7 727 Enhancer 4KVQLVESGGGLVQAGGSLRLSCAASGRT VSDYGMGWFRQAPGKLREFVASINWSGIYTRYIDSVEGRFTISRDNTKNTLYLQMNN LRAEDTAVYYCAYFLGPNWYSNYGRPSSYDFYGQGTQVTVSS P2X7PMP19A8 728 Enhancer 4 EVQLMESGGGLVQAGGSLRLSCAASGRTVSDYGMGWFRQAPGKLREFVASINWS GIYTRYIDSVEGRFTISRDNTKNTLYLQMNNLKAEDTAVYYCAYFLGPNWYSNYGRPS SYDFYGQGTQVTVSS P2X7PMP19D12 729 Enhancer 4EVQLVESGGGLVQAGGSLRLSCAASGRT VSDYGMGWFRQAPGKLREFVASINWSGIYTRYIDSVEGRFTISRDNTKNTLYLQMNN LKAEDTAVYYCAYFLGPNWYSNYGRPSSYGFYGQGTQVTVSS P2X7PMP20B8 730 Enhancer 4 EVQLVESGGGLVQAGGSLRLSCAASGRTVSDYGMGWFRQAPGKECEFVASINWSG TYTRYIDSVEGRFTISRDNTENTLYLQMNNLKAEDTAVYYCAYFLGPNWYSDYGRPS SYDFYGQGTQVTVSS P2X7PMP4G4 731 Enhancer 4EVQLMESGGGLVQAGGPLRLSCAASGR TVSDYGMGWFRQAPGKEREFVASINWSGTYTRYIDSVEGRFTISRDNTENTLYLQM NNLKAEDTAVYYCAYFLGPNWYSDYGRPSSYDFYGQGTQVTVSS P2X7PMP8G11 732 Blocker 5, 1AVQLVESGGGLVQAGGSLRLSCAASGNF FRVNTMAWYRQAPGKQRELVADITRGDRTNYADTVNGRFTISRDNVRNTVYLQMNG LRPEDTAAYYCYAVIELGVLEPRDYWGQ GTQVTVSSP2X7PMP6A11 733 Blocker 5, 2 EVQLVESGGGLVQAGGSLRLSCAASGNFFRVNTMAWYRQAPGKQRELVADITRGDR TNYADTVNGRFTISRDNVRNTVYLQMNGLKPEDTAAYYCYARIELGVLEPRDYWGQ GTQVTVSS P2X7PMP8E6 734 Blocker 5, 2EVQLVESGGGLVQAGGPLRLSCAASGNF FRVNTMAWYRQAPGKQRELVADITRGDRTNYADTVNGRFTISRDNVRNTVYLQMNG LKPEDTAAYYCYARIELGVLEPRDYWGQ GTQVTVSSP2X7PMP12A11 735 Blocker 5, 2 EVQLVESGGGLVQAGGSLRLSCAASGSFFRVNNMAWYRQAPGKQRELVADITRGD RTNYADSVNGRFTISRDNVRNTVYLQMNGLKPEDTAVYYCYARIELGVLEPRDYWG QGTQVTVSS P2X7PMP14F6 736 Blocker 5, 2EVQLVESGGGLVQAGGPLRLSCAASGSF FRVNNMAWYRQAPGKQRELVADITRGDRTNYADSVNGRFTISRDNVRNTVYLQMN GLKPEDTAVYYCYARIELGVLEPRDYWG QGTQVTVSSP2X7PMP8B4 737 Blocker 5, 2 EVQLVESGGGLVQAGGSLRLSCAASGNFFRVNTMAWYRQAPGKQRELVADITRGDR TNYADTVNGRFTISRDNVRNTVYLQMNSLKPEDTAAYYCYARIELGVLEPRDYWGQ GTQVTVSS P2X7PMP14G4 738 Blocker 5, 2EVQLVESGGGLVQAGGSLGLSCAASGNF FRVNTMAWYRQAPGKQRELVADITRGDRTNYADTVNGRFTISRDNVRNTVYLQMNG LKPEDTAAYYCYARIELGVLEPRDYWGQ GTQVTVSSP2X7PMP8H5 739 Blocker 5, 2 EMQLVESGGGLVQAGGSLRLSCAASGNFFRVNTMAWYRQAPGKQRELVADITRGD RTNYADTVNGRFTISRDNVRNTVYLQMNGLKPEDTAAYYCYARIELGVLEPRDYWG QGTQVTVSS P2X7PMP14F10 740 Blocker 5, 2EVQLVESGGGLVQAGGSLRLLCAASGSF FRVNNMAWYRQAPGKQRELVADITRGDRTNYADSVNGRFTISRDNVRNTVYLQMN GLKPEDTAVYSCYARIELGILEPRDYWGQ GTQVTVSSP2X7PMP8A11 741 Blocker 5, 2 EVQLVESGGGLVQAGGSLRLSCAASGSFFRVNNMAWYRQAPGKQRELVADITRGD RTNYADSVNGRFTISRDNVRNTVYLQMDGLKPEDTAVYYCYARIELGVLVPRDYWG QGTQVTVSS P2X7PMP8H6 742 Blocker 5, 2EVQLVESGGGLVQAGGSLRLSCAASGSF FRVNNMAWYRQAPGKQRELVADITRGDRTNYADSVNGRFTISRDNVRNTVYLQMN GLKPEDTAVYYCYARIELGPLVPRDYWG QGTQVTVSSP2X7PMP8F5 743 Blocker 5, 2 EVQLVKSGGGLVQAGGSLRLSCAASGSFFRVNNMAWYRQAPGKQRELVADITRGD RTNYADSVNGRFTISRDNVRNTVYLQMNGLKPEDTAVYYCYARIELGPLVPRDYWG QGTQVTVSS P2X7PMP8G12 744 Blocker 5, 3KVQLVESGGGLVQAGGSLRLSCAASGSF FRVNNMAWYRQGPGKQRELVADITRGDRTNYADSVNGRFTISRDNVRNTVYLQMN GLKPEDTAVYYCYATIELGVLEPRDYWG QGTQVTVSSP2X7PMP8B12 745 Blocker 5, 3 EVQLVESGGGLVQAGGSLRLSCAASGSFFRVNVMAWYRQGPGKQRELVADITRGD RTNYADSVNGRFTISRDNVRNTVYLQMNGLKPEDTAVYYCYATIELGVLEPRDYWG QGTQVTVSS P2X7PMP14G11 746 Blocker 5, 3EVQLVESGGGLVKPGGSLRLSCAASGSF FRVNNMAWYRQGPGKQRELVADITRGDRTNYADSVNGRFTISRDNVRNTVYLQMN GLKPEDTAVYYCYATIELGVLEPRDYWG QGTQVTVSSP2X7PMP8C12 747 Blocker 5, 3 EVQLVESGGGLVQAGGSLRLSCAASGSFFRVNNMAWYRQAPGKQRELVADITRGD RTNYADSVNGRFTISRDNVRNTVYLQMNGLKPEDTAVYYCYATIELGVLEPRDYWG QGTQVTVSS P2X7PMP8H10 748 Blocker 5, 3EVQLVESGGGLVQAGGSLRLSCAASGSF FRVNNMAWYRQGPGKQRELVADITRGDRTNYADSVNGRFTISRDNVRNTVYLQMN GLKPEDTAVYYCYATIELGVLEPRDYWG QGTQVTVSSP2X7PMP8D10 749 Blocker 5, 3 EVQLVESGGGLVQPGGSLRLSCAASGSFFRVNNMAWYRQAPGKQRELVADITRGD RTNYADSVNGRFTISRDNVRNTVYLQMNGLKPEDTAVYYCYATIELGVLEPRDYWG QGTQVTVSS P2X7PMP8H4 750 Blocker 5, 3EVQLVESGGGLVQAGGSLRLSCAASGSF FRVNNMAWYRQGPGKQRELVADITRGDRTNYADSVNGRFTISRDNVRNTVYLQMD GLKPEDTAVYYCYATIELGVLVPRDYWG QGTQVTVSSP2X7PMP18D12 751 Binder 6 EVQLVESGGDLVQAGGSLKLSCVVSGVTFDDGTIGWFRQAPGKEREGIACISRVDGT TYYRDSVKGRFTVSSDSAKTTVNLQMNSLKPEDTAVYYCAADYASLCTIETGYGSLY DYWGRGTQVTVSS P2X7PMP4B3 752 Binder 6EVQLVESGGDLVQAGGSLKLSCVVSGVT FDDGTIGWFRQAPGKEREGIACISRVDGTTYYRDSVKGRFTVSSDSAKTTVNLQMNS LKPEDTAVYYCAADYASLCTIETGYGSLY DYWGKGTQVTVSSP2X7PMP8C7 753 Partial 7 EMQLVESGGGLVQAGGSLRLSCAASGR BlockerTFSSLAMGWLRQAPGKEREFVSGISRGG TSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAGSPVLSIVLDTRGLE YWGQGTQVTVSS P2X7PMP8E7 754 Partial 7EVQLVESGGGLVQAGGSLRLSCAASGRT Blocker FSSLAMGWLRQAPGKEREFVSGISRGGTSTYYADSVKGRFTISRDNAKNTMYLQMN SLKPEDTAVYYCAGSPVLSIVLDTRGLEY WGQGTQVTVSSP2X7PMP6D7 755 Partial 7 EVQLVESGGGLVQAGGSLRLSCAASGRT BlockerFSSLAMGWLRQAPGKEREFVSGISRGGT STYYADSVKGRLTISRDNAKNTMYLQMNSLKPEDTAVYYCAGSPVLSIVLDTRGLEY WGQGTQVTVSS P2X7PMP7D6 756 Enhancer 8EVQLVESGGGLVQAGGSLRLSCAASGRT FGSSPVGWFRQAPGKERDFVATISWNGVDTHYLDSVKGRFTISRDNAKNTVHLQM HILKPEDTALYYCAASTSGSVYLPYRVYQYDSWGQGTQVTVSS P2X7PMP7E8 757 Enhancer 8 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSSPVGWFRQAPGKERDFVATISWNGV DTHYLDSVKGRFTISRDNALNTVHLQMHILKPEDTALYYCAASTSGSAYLPYRVYQYD SWGQGTQVTVSS P2X7PMP7F5 758 Enhancer 8EVQLVESGGGLVQAGGSLRLSCAASGRT FSSSPVGWFRQAPGKERDFVATISWNGVDTHYLDSVKGRFTISRDNALNTVHLQMHI LKPEDTALYYCAASTSGSAYLPYRVHQY DSWGQGTQVTVSSP2X7PMP7F9 759 Binder 9 EVQLVESGGGLVQAGASLRVSCAASARTGSYTMGWFRQAPGKEREFVSTISWNGA STVYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCAGSISSYSSRWQDDYE YWGQGTQVTVSS P2X7PMP7A4 760 Binder 9EVQLVESGGGLVQAGGSLRVSCAASART GSYSMGWFRQAPGKEREFVSTISWNGADTVYADSVKGRFTISRDNAKDTVYLQMN SLKPEDTAVYYCAGSITSYVSTWQHDYE YWGQGTQVTVSSP2X7PMP7B4 761 Binder 10 EVQLVESGGGLVQAGGSLRLSCAASGRNFGSYTMAWFRQAPGKGREFVSTINWSG GDTDYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAAGLEYMSTIRYTYEY WGQGTQVTVSS P2X7PMP7H6 762 Blocker 11EVQLVESGGGLVQPGGSLRLSCVVSGS MYRIDNMGWYRQAPGKQRELVATVTRGDITNYADSVKGRFTIGRDNAKNTVYLQMN SLKPADTAVYYCNIDSYIIGAGVRDYWGR GTQVTVSSP2X7PMP7G5 763 Enhancer 12 EVQLVESGGGLVQSGGSLRLSCAGSGFSYYIIGWFRQAPGKEREEVSCIRVTDGSTY YTNSVKGRFTMSRDNAENTVYLQMNSLKPEDTAVYSCATECQRWAYPNRIGARGQ GTQVTVSS P2X7PMP13F4 764 Enhancer 12EVQLVESGGGLVQSGGSLRLSCAGSGFS YYIIGWFRQAPGKEREEVSCIRVTDGSTYHTNSVKGRFTMSRDNAENTVYLQMNSLK PEDTAVYSCATECQRWAYPNRIGARGQ GTQVTVSSP2X7PMP7D8 765 Binder 13 EVQLVESGGGLVQPGGSLRLSCAASGLTLEYYNIGWFRQAPGKEREGVACIDWTEG STFYVDSVKGRFTISTDNAKNTVYLHMNSLEPEDTAVYYCAAGWGRVITVQHMCADR SLFTSWGQGTQVTVSS P2X7PMP13B8 766 Binder 13EVQLVESGGGLVQPGGSLRLSCAASGLT LTYYNIGWFRQAPGKEREGVSCIDWTDGTTFYADSVKGRFTISTDNAKNTVYLHLNS LEPEDTAVYYCAAGWGRVMTVQHMCADRSLFTSWGQGTQVTVSS P2X7PMP7D5 767 Binder 14 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSVAVGWFRQAPGKERDFVAWISWSG DSTYYADSVKGRFTASRDNVNNTVYLQMNSLKPEDTADYYCAAAWKYDRASYDFPE AYDYWGQGTQVTVSS P2X7PMP16D9 768 Binder 15EVQLVESGGGLVQAGGSLRLSCAASPGT FSSFNMGWFRQTPGKEREFVAATSWSDISTYYADSVKGRFTISRDNAKNTVTLEMNS LKPEDTAVYYCAAGYYRGGYLGYRLTLEGSYDVWGQGTQVTVSS P2X7PMP1G6 769 Binder 15 EVQLVESGGGLVQAGGSLRLSCAASPGAFSSFNMGWFRQTPGKEREFVAATSWSDI STYYADSVKGRFTISRDNAKNTVTLEMNSLKPEDTAVYYCAAGYYRGGYLGYRLTLE GSYDVWGQGTQVTVSS P2X7PMP19E3 770 Binder 15EMQLVESGGGLVQAGGSLRLSCAASPGT FSSFNMGWFRQTPGKEREFVAATSWSDISTYYADSVKGRFTISRDNAKNTVTLEMNS LKPEDTAVYYCAAGYYRGGYLGYRLTLEGSYDVWGQGTQVTVSS P2X7PMP19C2 771 Binder 15 EVQLVKSGGGLVQAGGSLRLSCAASPGTFSSFNMGWFRQTPGKEREFVAATSWSDI STYYADSVKGRFTISRDNAKNTVTLEMNSLKPEDTAVYYCAAGYYRGGYLGYRLTLE GSYDVWGQGTQVTVSS P2X7PMP6B7 772 Partial 16EVQLVESGGGLVQAGGSLRLSCVVSGRT Blocker FSAMGWFRQAPGKEREFVATVGWNPMNSYYGDSVKGRFTIFRDNAKNTVYLQMNS LKPEDTAVYYCAGSGSLLDVTSEAVYTD WGQGTQVTVSSP2X7PMP14D5 773 Enhancer 17 KVQLVESGGGLVQAGGSLRLSCAASGSPISSYAMGWYRQAPGKPRELVARIYTGGT AWYEDSVKGRFTISRDNAQNTVYLQMNSLKSEDTAVYYCHGRVRYDYWGQGTQVT VSS P2X7PMP13G5 774 Binder 18EVQLVESGGGLVQAGGSLRLSCAASDRT FGSSAMGWFRQAPGKDRDFVAAISWSGSSTHYADSVKGRFTISRDNAKNTMYLQM NSLKPADTAVYTCAASRRAYLPAKVGEY DFWGQGTQVTVSSP2X7PMP13B5 775 Partial 19 EVQLVESGGGLVQAGDSLRLSCAASGRT BlockerFSSYAMGWFRQAPGKEREFVAAISLSGS MTYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEELGDGLGYLAYRY DYWGQGTQVTVSS P2X7PMP13F6 776 Binder 20EVQLVESGGGLVQPGGSLRLSCAASGFT FSTNIMTWVRQAPGKGLEWISTINSGGGTTTYADSVRGRFTISRDNAKNMLYLQMSSL KPEDTALYYCITPRGVKGRGTQVTVSS P2X7PMP13G4777 Binder 21 EVQLVESGGGLVQAGGSLRLSCAASDRT FGSSTMGWFRQPPGKNREFVATIAWSATTTHYADAVKGRFTVSRDNALNTVYLQMN SLKPEDTAVYYCAATLTWLGIHEYEYNTW GQGTQVTVSSP2X7PMP13A7 778 Blocker 22 EVQLVESGGGLVQPGESLRLSCTASRFMLDYYDIGWFRQAPGKEREGVSCRFTNDG STAYADSVKGRFTISRDIVKHTVYLQMNSLQPEDTAVYYCAAGPLTKRRQCVPGDFS MDFWGEGTLVTVSS P2X7PMP13E9 779 Blocker 22EVQLVESGGGLVQPGESLRLSCTASRFD LDYYDIAWFRQAPGKEREGVSCSFTNDGSTYYADSVKGRFTMSRNNDHRTVYLQMT SLQPEDTAVYTCAVGPLTRRRQCVPGDFSMDFWGEGTLVTVSS P2X7PMP13G9 780 Blocker 23 EVQLVESGGGLVQAGGSLRLSCVASGRTFSILTMGWFRQAPGKEREFVAAISGIGAIH YADSVKGRFTLSRDNARNTVSLHMNSLKPEDTAVYYCAAKANYESPSRETSYAYWG QGTQVTVSS P2X7PMP20H9 781 Binder 24EVQLVESGGRVMQTGGSLRLSCAASGH TFNDYSMGWFRQAPGKELEFLAGINWSGMSTWYADSVKDRFTISRDNNKNTVFLQM NSLEPGDTAVYYCAARQWISTIILTAPSQYDYWGQGTQVTVSS P2X7PMP20A11 782 Binder 24 EVQLVESGGRVMQTGGSLRLSCAASGHTFNDYNMGWFRQAPGKELEFLAGINWS GMSTWYADSVKDRFTISRDNNKNTVFLQMNSLEPGDTAVYYCAARQWISTIILTAPS QYDYWGQGTQVTVSS P2X7PMP18C1 783 Binder 25EVQLVESGGDLVQPGGSLRLSCVASGFA LEEHAIGWFRQAPGKEREGVSLSSYLGAAYYATSVKGRFTISRDNAKNTVTLQMNSL KPEDTAVYYCARGHFTYDDGRITIRSVDY WGKGTLVTVSSP2X7PMP18A7 784 Binder 25 EVQLVESGGDLVQPGGSLRLSCVASGFALEEHAIGWFRQAPGKEREGVSLSSYVGA VYYATSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCARGHFTYDDGRISIRSVDH WGKGTLVTVSS P2X7PMP19H4 785 Binder 26EVQLVESGGGLVQAGGSLRLSCVASGRT LSTAVMGWFRQAPGKERGLVAMISWSGSMTYYAKSVKGRFTISRDNAKNTMYLQM NSLKPEDTAVYYCAADMGGGPPDGDAMPRLSSGMDYWGKGTLVTVSS P2X7PMP16G3 786 Binder 27EVQLVESGGGLVQAGGSLRLSCAASGND FARFSIDAMGWYRQAPGKQRELVATVTEDGTKNYADSVKGRATISRDDANNSMYLE MNTLKPEDTAVYYCKMGGLIDGAAPYEF WGRGTQVTVSSP2X7PMP15C7 787 Binder 28 EMQLVESGGGWVQAGGSLRLSCASSGSIFSAGAMGWYRQPAGKQRELVADITLGG STNYADSVKGRFTISRDNAKNAVFLQMNSLKPEDTAVYYCNGLINTFARKIPRYAWG QGTQVTVSS P2X7PMP16F5 788 Binder 29EVQLVESGGGLVQAGGSLRLSCAASGPT TFGRYTMGWFRQAPGREREFVAAISWIGGRTYYVDVVKGRFTISRDNAKKMVYLQM NSLKPDDTAVYHCAAAFQALGSPREYDY WGQGTQVTVSS

In particular, the invention in some specific aspects provides:

-   -   immunoglobulin sequences that are directed against (as defined        herein) an ion channel and that have at least 80%, preferably at        least 85%, such as 90% or 95% or more sequence identity with at        least one of the immunoglobulin sequences of SEQ ID NO's: 705 to        788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to        764, 772 to 773, 775, or 778 to 780, more preferably SEQ ID NO's        726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or 778 to        780 (see Table A-1). These immunoglobulin sequences may further        be such that they neutralize binding of the cognate ligand to an        ion channel; and/or compete with the cognate ligand for binding        to an ion channel; and/or are directed against an interaction        site (as defined herein) on an ion channel (such as the ligand        binding site);    -   immunoglobulin sequences that cross-block (as defined herein)        the binding of at least one of the immunoglobulin sequences of        SEQ ID NO's: 705 to 788, more preferably SEQ ID NO's 726 to 750,        753 to 758, 762 to 764, 772 to 773, 775, or 778 to 780, more        preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772        to 773, 775, or 778 to 780 (see Table A-1) to an ion channel        and/or that compete with at least one of the immunoglobulin        sequences of SEQ ID NO's: 705 to 788, more preferably SEQ ID        NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or 778        to 780, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762        to 764, 772 to 773, 775, or 778 to 780 (see Table A-1) for        binding to an ion channel. Again, these immunoglobulin sequences        may further be such that they neutralize binding of the cognate        ligand to an ion channel; and/or compete with the cognate ligand        for binding to an ion channel; and/or are directed against an        interaction site (as defined herein) on an ion channel (such as        the ligand binding site);        which immunoglobulin sequences may be as further described        herein (and may for example be Nanobodies); as well as        polypeptides of the invention that comprise one or more of such        immunoglobulin sequences (which may be as further described        herein, and may for example be bispecific and/or biparatopic        polypeptides as described herein), and nucleic acid sequences        that encode such immunoglobulin sequences and polypeptides. Such        immunoglobulin sequences and polypeptides do not include any        naturally occurring ligands.

Accordingly, some particularly preferred Nanobodies of the invention areNanobodies which can bind (as further defined herein) to and/or aredirected against to an ion channel and which:

-   i) have at least 80% amino acid identity with at least one of the    immunoglobulin sequences of SEQ ID NO's: 705 to 788, more preferably    SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or    778 to 780, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762    to 764, 772 to 773, 775, or 778 to 780 (see Table A-1), in which for    the purposes of determining the degree of amino acid identity, the    amino acid residues that form the CDR sequences are disregarded. In    this respect, reference is also made to Table B-1, which lists the    framework 1 sequences (SEQ ID NO's: 126 to 207), framework 2    sequences (SEQ ID NO's: 290 to 371), framework 3 sequences (SEQ ID    NO's: 454 to 535) and framework 4 sequences (SEQ ID NO's: 618    to 699) of the Nanobodies of SEQ ID NO's: 705 to 788, more    preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to    773, 775, or 778 to 780, more preferably SEQ ID NO's 726 to 750, 753    to 758, 762 to 764, 772 to 773, 775, or 778 to 780 (see Table A-1)    (with respect to the amino acid residues at positions 1 to 4 and 27    to 30 of the framework 1 sequences, reference is also made to the    comments made below. Thus, for determining the degree of amino acid    identity, these residues are preferably disregarded);    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 below.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Again, such Nanobodies may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic immunoglobulin sequences, including but not limited to“humanized” (as defined herein) Nanobodies, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a Nanobodycomprises a V_(HH) sequence, said Nanobody may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized Nanobodies of the invention. Similarly,when a Nanobody comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said Nanobody may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

In particular, humanized Nanobodies may be immunoglobulin sequences thatare as generally defined for Nanobodies in the previous paragraphs, butin which at least one amino acid residue is present (and in particular,in at least one of the framework residues) that is and/or thatcorresponds to a humanizing substitution (as defined herein). Somepreferred, but non-limiting humanizing substitutions (and suitablecombinations thereof) will become clear to the skilled person based onthe disclosure herein. In addition, or alternatively, other potentiallyuseful humanizing substitutions can be ascertained by comparing thesequence of the framework regions of a naturally occurring V_(HH)sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized V_(HH) sequences can be tested for affinity for the target,for stability, for ease and level of expression, and/or for otherdesired properties. In this way, by means of a limited degree of trialand error, other suitable humanizing substitutions (or suitablecombinations thereof) can be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) a Nanobody may be partially humanized or fully humanized.

In a preferred but non-limiting aspect, the invention relates to aNanobody (as defined herein) against ion channels such as e.g. P2X7,which consists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

-   -   CDR1 is chosen from the group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;        and/or    -   CDR2 is chosen from the group consisting of:    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;        and/or    -   CDR3 is chosen from the group consisting of:    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   i) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;        or any suitable fragment of such an immunoglobulin sequence.

In particular, according to this preferred but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) against ion channelssuch as e.g. P2X7, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which:

-   -   CDR1 is chosen from the group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;        and    -   CDR2 is chosen from the group consisting of:    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;        and    -   CDR3 is chosen from the group consisting of:    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;

i) immunoglobulin sequences that have 3, 2, or 1 amino acid differencewith at least one of the immunoglobulin sequences of SEQ ID NO's: 536 to617;

or any suitable fragment of such an immunoglobulin sequences.

As generally mentioned herein for the immunoglobulin sequences of theinvention, when a Nanobody of the invention contains one or more CDR1sequences according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)    and/or c) is preferably, and compared to the corresponding CDR    according to a), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to b) and/or c) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to a);    and/or-   iii) the CDR according to b) and/or c) may be a CDR that is derived    from a CDR according to a) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Similarly, when a Nanobody of the invention contains one or more CDR2sequences according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)    and/or f) is preferably, and compared to the corresponding CDR    according to d), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to e) and/or f) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to d);    and/or-   iii) the CDR according to e) and/or f) may be a CDR that is derived    from a CDR according to d) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Also, similarly, when a Nanobody of the invention contains one or moreCDR3 sequences according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)    and/or i) is preferably, and compared to the corresponding CDR    according to g), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to h) and/or i) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to g);    and/or-   iii) the CDR according to h) and/or i) may be a CDR that is derived    from a CDR according to g) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally applyto any Nanobody of the invention that comprises one or more CDR1sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e),f), h) or i), respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more ofthe CDR's explicitly listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and Nanobodies comprising three of the CDR'sexplicitly listed above are most particularly preferred.

-   -   Some particularly preferred, but non-limiting combinations of        CDR sequences, as well as preferred combinations of CDR        sequences and framework sequences, are mentioned in Table B-1        below, which lists the CDR sequences and framework sequences        that are present in a number of preferred (but non-limiting)        Nanobodies of the invention. As will be clear to the skilled        person, a combination of CDR1, CDR2 and CDR3 sequences that        occur in the same clone (i.e. CDR1, CDR2 and CDR3 sequences that        are mentioned on the same line in Table B-1) will usually be        preferred (although the invention in its broadest sense is not        limited thereto, and also comprises other suitable combinations        of the CDR sequences mentioned in Table B-1). Also, a        combination of CDR sequences and framework sequences that occur        in the same clone (i.e. CDR sequences and framework sequences        that are mentioned on the same line in Table B-1) will usually        be preferred (although the invention in its broadest sense is        not limited thereto, and also comprises other suitable        combinations of the CDR sequences and framework sequences        mentioned in Table B-1, as well as combinations of such CDR        sequences and other suitable framework sequences, e.g. as        further described herein).    -   Also, in the Nanobodies of the invention that comprise the        combinations of CDR's mentioned in Table B-1, each CDR can be        replaced by a CDR chosen from the group consisting of        immunoglobulin sequences that have at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity (as defined herein) with the        mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table B-1, a    conservative amino acid substitution (as defined herein);    and/or

-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table B-1;    and/or

-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table B-1.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table B-1 will generally be preferred.

TABLE B-1 Preferred combinations of CDR sequences, preferredcombinations of framework sequences, and preferred combinations offramework and CDR sequences. (“ID” refers to the SEQ ID NO as usedherein) ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 126 EVQLVESG208 DYAIG 290 WFRQAPGKE 372 CISSTGNVFYADSVKG 454 RFTISSDKEKNTLYLQ 536GHFTVDSGKV 618 WGQGT GDVVQAGG REGIS MNSLKPEDTAVYHCAA LLRTDISS QVTVSSSLRLSCLAS GFTFD 127 EVQLVESG 209 DYAIG 291 WFRQAPGKE 373CISSTGNVFYADSVKG 455 RFTISSDKEKNTLYLQ 537 GHFTVDSGKV 619 WGQGT GDVVQAGGREGIS MNSLEPEDTAVYHCAA LLRTDISS QVTVSS SLRLSCLAS GFTFD 128 EMQLVESG 210DYAIG 292 WFRQAPGKE 374 CISSTGNVFYADSVKG 456 RFTISSDKEKNTLYLQ 538GHFTVDSGKV 620 WGQGT GDVVQAGG REGIS MNSLKPEDTAVYHCAA LLRTDISS QVTVSSSLRLSCLAS GFTFD 129 EVQLVESG 211 DYAIG 293 WFRQAPGKE 375CISSTGNVFYADSVKG 457 RFTISSDKEKNTLYLQ 539 GHFTVDSGKV 621 WGQGT GDVVQAGGREGIS MNSLKPGDTAVYHC LLRTDISS QVTVSS SLRLSCLAS AA GFTFD 130 EVQLVESG 212DYAIG 294 WFRQAPGKE 376 CISSTGNVFYADSVKG 458 RLTISSDKEKNTLYLQ 540GHFTVDSGKV 622 WGQGT GDVVQAGG REGIS MNSLKPEDTAVYHCAA LLRTDISS QVTVSSSLRLSCLAS GFTFD 131 EVQLVESG 213 DYAIG 295 WFRQAPGKE 377CISSTGNVFYADSVKG 459 RFTISSDKEKNTLYLQ 541 GHFTVDSGKV 623 WGQGT GDVVQAGGREGIS MDSLKPEDTAVYHCAA LLRTDISS QVTVSS SLRLSCLAS GFTFD 132 EVQLVESG 214DYAIG 296 WFRQAPGKE 378 CISSTGNVFYADSVKG 460 RFTISSDKEKNTLYLQ 542GHFTVDSGKV 624 WGQGT GDVVQAGG REGIS MNSLKPEDTAVYHCAA LLRTDVSS QVTVSSSLRLSCLAS GFTFD 133 EVQLVESG 215 DYAIA 297 WFRQAPGKE 379ILSSIGKTFYADSVKD 461 RFSITADGAKTTVFLQ 543 GHFVYNDGAI 625 WGQGA GGLVQTGGREGVS MNSLKPGDTAIYYCVA SLNTARGSGF QVTVSS SLRLSCAAS GFTLD 134 EVQLVESG216 DYAIA 298 WFRQAPGKE 380 ILSSIGKTFYADSVKD 462 RFSITADGAKTTVFLQ 544GHFVYNDGAI 626 WGQGA GGLVQTGG REGVS MNSLKPEDTAIYYCVA SLNTARGSGF QVTVSSSLRLSCAAS GFTLD 135 EVQLVESG 217 DYAIA 299 WFRQAPGKE 381ILSSIGKTFYADSVKD 463 RFSITADGAKTTVFLQ 545 GHFVYNDGAI 627 WGQGT GGLVQTGGREGVS MNSLKPEDTAIYYCVA SLNTARGSGF QVTVSS SLRLSCAAS GFTLD 136 EVQLVESG218 DYAIA 300 WFRQAPGKE 382 ILSSIGKTFYADSAKD 464 RFSITADGAKTTVFLQ 546GHFVYNDGAI 628 WGQGT GGLVQTGG REGVS MNSLKPEDTAIYYCVA SLNTARGSGF QVTVSSSLRLSCAAS GFTLD 137 EVQLVESG 219 MG 301 WFRQAPGKE 383 GSGWDGIPTRYADSVKG465 RLTISRDNAKNTVSL 547 GTSVYHYQY 629 WGQGT GGLVQAGG REFVAQMSGLKPEDTAIYYC QVTVSS SLRLSCAAS AT ERTYS 138 EVQLVESG 220 MG 302WFRRAPGKE 384 GSGWDGIPTRYADSVKG 466 RFTISRDNAKNTVSL 548 GTSVYHYQY 630WGQGT GGLVQAGG REFVA QMSGLKPEDTAIYYC QVTVSS SLRLSCAAS AT ERTYS 139EVQLVESG 221 VG 303 WFRQAPGKE 385 GSGWDGTPTRYADSVKG 467 RFTISRDNAKNTVSL549 GTSVYHYQY 631 WGQGT GGLVQAGG REFVA QMSGLKPEDTAIYYC QVTVSS SLRLSCAASAT ERTYS 140 EVQLVESG 222 MG 304 WFRQAPGKE 386 GSGWDGIPTRYADSVKG 468RFTISRDNAKNTVSL 550 GTSVYHYQY 632 WGQGT GGLVQAGG REFVA QMSGLKPEDTAIYYCQVTVSS SLRLSCAAP AT ERTYS 141 EVQLVESG 223 MG 305 WFRQAPGKE 387GSGWDGIPTRYADSVKG 469 RFTISRDNAKNTVSL 551 GTSVYHYQY 633 WGQGT GGLVQAGGREFVA QMSGLKPEDTAIYYC QVTVSS SLRLSCAAS AT ERTYS 142 EVQLVESR 224 MG 306WFRQAPGKE 388 GSGWDGIPTRYADSVKS 470 RFTISRDNAKNTVSL 552 GTSVYHYQY 634WGQGT GGLVQAGG REFVA QMSGLKPEDTAIYYC QVTVSS SLRLSCAAS AT ERTYS 143EVQLVESG 225 MG 307 WFRQAPGKE 389 GSGWDGIPTRYADSVKG 471 RFTISRDNAKNAVSL553 GTSVYHYQY 635 WGQGT GGLVQAGG REFVA QMSGLKPEDTAIYYC QVTVSS SLRLSCAASAT ERTYS 144 EVQLVESG 226 MG 308 WFRQAPGKE 390 GSGWDGIPTRYADSVKG 472RFTISRDNAKNTVSL 554 GTSVYHYQY 636 WGQGT GGLVQPGG REFVA QMSGLKPEDTAIYYCQVTVSS SLRLSCAAS AT ERTYS 145 EVQLVESG 227 MG 309 WFRQAPGKE 391GSGWDGIPTRYADSVKG 473 RFTISRDNAKSTVSL 555 GTSVYHYQY 637 WGQGT GGLVQAGGREFVA QMSGLKPEDTAIYYC QVTVSS SLRLSCAAS AT ERTYS 146 KVQLVESG 228 DYGMG310 WFRQAPGKL 392 SINWSGIYTRYIDSVEG 474 RFTISRDNTKNTLYLQ 556 FLGPNWYSN638 YGQGTQ GGLVQAGG REFVA MNNLRAEDTAVYYC YGRPSSYDF VTVSS SLRLSCAAS AYGRTVS 147 EVQLMESG 229 DYGMG 311 WFRQAPGKL 393 SINWSGIYTRYIDSVEG 475RFTISRDNTKNTLYLQ 557 FLGPNWYSN 639 YGQGTQ GGLVQAGG REFVAMNNLKAEDTAVYYCAY YGRPSSYDF VTVSS SLRLSCAAS GRTVS 148 EVQLVESG 230 DYGMG312 WFRQAPGKL 394 SINWSGIYTRYIDSVEG 476 RFTISRDNTKNTLYLQ 558 FLGPNWYSN640 YGQGTQ GGLVQAGG REFVA MNNLKAEDTAVYYCAY YGRPSSYGF VTVSS SLRLSCAASGRTVS 149 EVQLVESG 231 DYGMG 313 WFRQAPGKE 395 SINWSGTYTRYIDSVEG 477RFTISRDNTENTLYLQ 559 FLGPNWYSD 641 YGQGTQ GGLVQAGG CEFVAMNNLKAEDTAVYYCAY YGRPSSYDF VTVSS SLRLSCAAS GRTVS 150 EVQLMESG 232 DYGMG314 WFRQAPGKE 396 SINWSGTYTRYIDSVEG 478 RFTISRDNTENTLYLQ 560 FLGPNWYSD642 YGQGTQ GGLVQAGG REFVA MNNLKAEDTAVYYCAY YGRPSSYDF VTVSS PLRLSCAASGRTVS 151 AVQLVESG 233 VNTMA 315 WYRQAPGKQ 397 DITRGDRTNYADTVNG 479RFTISRDNVRNTVYL 561 VIELGVLEPRDY 643 WGQGT GGLVQAGG RELVA QMNGLRPEDTAAYYQVTVSS SLRLSCAAS CYA GNFFR 152 EVQLVESG 234 VNTMA 316 WYRQAPGKQ 398DITRGDRTNYADTVNG 480 RFTISRDNVRNTVYL 562 RIELGVLEPR 644 WGQGT GGLVQAGGRELVA QMNGLKPEDTAAYY DY QVTVSS SLRLSCAAS CYA GNFFR 153 EVQLVESG 235VNTMA 317 WYRQAPGKQ 399 DITRGDRTNYADTVNG 481 RFTISRDNVRNTVYL 563RIELGVLEPR 645 WGQGT GGLVQAGG RELVA QMNGLKPEDTAAYY DY QVTVSS PLRLSCAASCYA GNFFR 154 EVQLVESG 236 VNNMA 318 WYRQAPGKQ 400 DITRGDRTNYADSVNG 482RFTISRDNVRNTVYL 564 RIELGVLEPR 646 WGQGT GGLVQAGG RELVA QMNGLKPEDTAVYYDY QVTVSS SLRLSCAAS CYA GSFFR 155 EVQLVESG 237 VNNMA 319 WYRQAPGKQ 401DITRGDRTNYADSVNG 483 RFTISRDNVRNTVYL 565 RIELGVLEPR 647 WGQGT GGLVQAGGRELVA QMNGLKPEDTAVYY DY QVTVSS PLRLSCAAS CYA GSFFR 156 EVQLVESG 238VNTMA 320 WYRQAPGKQ 402 DITRGDRTNYADTVNG 484 RFTISRDNVRNTVYL 566RIELGVLEPR 648 WGQGT GGLVQAGG RELVA QMNSLKPEDTAAYY DY QVTVSS SLRLSCAASCYA GNFFR 157 EVQLVESG 239 VNTMA 321 WYRQAPGKQ 403 DITRGDRTNYADTVNG 485RFTISRDNVRNTVYL 567 RIELGVLEPR 649 WGQGT GGLVQAGG RELVA QMNGLKPEDTAAYYDY QVTVSS SLGLSCAAS CYA GNFFR 158 EMQLVESG 240 VNTMA 322 WYRQAPGKQ 404DITRGDRTNYADTVNG 486 RFTISRDNVRNTVYL 568 RIELGVLEPR 650 WGQGT GGLVQAGGRELVA QMNGLKPEDTAAYY DY QVTVSS SLRLSCAAS CYA GNFFR 159 EVQLVESG 241VNNMA 323 WYRQAPGKQ 405 DITRGDRTNYADSVNG 487 RFTISRDNVRNTVYL 569RIELGILEPRDY 651 WGQGT GGLVQAGG RELVA QMNGLKPEDTAVYS QVTVSS SLRLLCAASCYA GSFFR 160 EVQLVESG 242 VNNMA 324 WYRQAPGKQ 406 DITRGDRTNYADSVNG 488RFTISRDNVRNTVYL 570 RIELGVLVPR 652 WGQGT GGLVQAGG RELVA QMDGLKPEDTAVYYDY QVTVSS SLRLSCAAS CYA GSFFR 161 EVQLVESG 243 VNNMA 325 WYRQAPGKQ 407DITRGDRTNYADSVNG 489 RFTISRDNVRNTVYL 571 RIELGPLVPR 653 WGQGT GGLVQAGGRELVA QMNGLKPEDTAVYY DY QVTVSS SLRLSCAAS CYA GSFFR 162 EVQLVKSG 244VNNMA 326 WYRQAPGKQ 408 DITRGDRTNYADSVNG 490 RFTISRDNVRNTVYL 572RIELGPLVPR 654 WGQGT GGLVQAGG RELVA QMNGLKPEDTAVYY DY QVTVSS SLRLSCAASCYA GSFFR 163 KVQLVESG 245 VNNMA 327 WYRQGPGKQ 409 DITRGDRTNYADSVNG 491RFTISRDNVRNTVYL 573 TIELGVLEPRDY 655 WGQGT GGLVQAGG RELVA QMNGLKPEDTAVYYQVTVSS SLRLSCAAS CYA GSFFR 164 EVQLVESG 246 VNVMA 328 WYRQGPGKQ 410DITRGDRTNYADSVNG 492 RFTISRDNVRNTVYL 574 TIELGVLEPRDY 656 WGQGT GGLVQAGGRELVA QMNGLKPEDTAVYY QVTVSS SLRLSCAAS CYA GSFFR 165 EVQLVESG 247 VNNMA329 WYRQGPGKQ 411 DITRGDRTNYADSVNG 493 RFTISRDNVRNTVYL 575 TIELGVLEPRDY657 WGQGT GGLVKPGG RELVA QMNGLKPEDTAVYY QVTVSS SLRLSCAAS CYA GSFFR 166EVQLVESG 248 VNNMA 330 WYRQAPGKQ 412 DITRGDRTNYADSVNG 494RFTISRDNVRNTVYL 576 TIELGVLEPRDY 658 WGQGT GGLVQAGG RELVA QMNGLKPEDTAVYYQVTVSS SLRLSCAAS CYA GSFFR 167 EVQLVESG 249 VNNMA 331 WYRQGPGKQ 413DITRGDRTNYADSVNG 495 RFTISRDNVRNTVYL 577 TIELGVLEPRDY 659 WGQGT GGLVQAGGRELVA QMNGLKPEDTAVYY QVTVSS SLRLSCAAS CYA GSFFR 168 EVQLVESG 250 VNNMA332 WYRQAPGKQ 414 DITRGDRTNYADSVNG 496 RFTISRDNVRNTVYL 578 TIELGVLEPRDY660 WGQGT GGLVQPGG RELVA QMNGLKPEDTAVYY QVTVSS SLRLSCAAS CYA GSFFR 169EVQLVESG 251 VNNMA 333 WYRQGPGKQ 415 DITRGDRTNYADSVNG 497RFTISRDNVRNTVYL 579 TIELGVLVPRDY 661 WGQGT GGLVQAGG RELVA QMDGLKPEDTAVYYQVTVSS SLRLSCAAS CYA GSFFR 170 EVQLVESG 252 DGTIG 334 WFRQAPGKE 416CISRVDGTTYYRDSVKG 498 RFTVSSDSAKTTVNL 580 DYASLCTIETG 662 WGRGT GDLVQAGGREGIA QMNSLKPEDTAVYY YGSLYDY QVTVSS SLKLSCVVS CAA GVTFD 171 EVQLVESG 253DGTIG 334 WFRQAPGKE 417 CISRVDGTTYYRDSVKG 499 RFTVSSDSAKTTVNL 581DYASLCTIETG 663 WGKGT GDLVQAGG REGIA QMNSLKPEDTAVYY YGSLYDY QVTVSSSLKLSCVVS CAA GVTFD 172 EMQLVESG 254 SLAMG 336 WLRQAPGKE 418GISRGGTSTYYADSVKG 500 RFTISRDNAKNTMYL 582 SPVLSIVLDTR 664 WGQGT GGLVQAGGREFVS QMNSLKPEDTAVYY GLEY QVTVSS SLRLSCAAS CAG GRTFS 173 EVQLVESG 255SLAMG 337 WLRQAPGKE 419 GISRGGTSTYYADSVKG 501 RFTISRDNAKNTMYL 583SPVLSIVLDTR 665 WGQGT GGLVQAGG REFVS QMNSLKPEDTAVYY GLEY QVTVSSSLRLSCAAS CAG GRTFS 174 EVQLVESG 256 SLAMG 338 WLRQAPGKE 420GISRGGTSTYYADSVKG 502 RLTISRDNAKNTMYL 584 SPVLSIVLDTR 666 WGQGT GGLVQAGGREFVS QMNSLKPEDTAVYY GLEY QVTVSS SLRLSCAAS CAG GRTFS 175 EVQLVESG 257SSPVG 339 WFRQAPGKE 421 TISWNGVDTHYLDSVKG 503 RFTISRDNAKNTVHL 585STSGSVYLPY 667 WGQGT GGLVQAGG RDFVA QMHILKPEDTALYYC RVYQYDS QVTVSSSLRLSCAAS AA GRTFG 176 EVQLVESG 258 SSPVG 340 WFRQAPGKE 422TISWNGVDTHYLDSVKG 504 RFTISRDNALNTVHL 586 STSGSAYLPY 668 WGQGT GGLVQAGGRDFVA QMHILKPEDTALYYC RVYQYDS QVTVSS SLRLSCAAS AA GRTFS 177 EVQLVESG 259SSPVG 341 WFRQAPGKE 423 TISWNGVDTHYLDSVKG 505 RFTISRDNALNTVHL 587STSGSAYLPY 669 WGQGT GGLVQAGG RDFVA QMHILKPEDTALYYC RVHQYDS QVTVSSSLRLSCAAS AA GRTFS 178 EVQLVESG 260 YTMG 342 WFRQAPGKE 424TISWNGASTVYADSVKG 506 RFTISRDNAKNTVSL 588 SISSYSSRWQ 670 WGQGT GGLVQAGAREFVS QMNSLKPEDTAVYY DDYEY QVTVSS SLRVSCAAS CAG ARTGS 179 EVQLVESG 261YSMG 343 WFRQAPGKE 425 TISWNGADTVYADSVKG 507 RFTISRDNAKDTVYL 589SITSYVSTWQ 671 WGQGT GGLVQAGG REFVS QMNSLKPEDTAVYY HDYEY QVTVSSSLRVSCAAS CAG ARTGS 180 EVQLVESG 262 SYTMA 344 WFRQAPGKG 426TINWSGGDTDYADSVKG 508 RFTISRDNAKNTVYL 590 GLEYMSTIRY 672 WGQGT GGLVQAGGREFVS QMDSLKPEDTAVYY TYEY QVTVSS SLRLSCAAS CAA GRNFG 181 EVQLVESG 263IDNMG 345 WYRQAPGKQ 427 TVTRGDITNYADSVKG 509 RFTIGRDNAKNTVYL 591DSYIIGAGVRDY 673 WGRGT GGLVQPGG RELVA QMNSLKPADTAVYY QVTVSS SLRLSCVVSCNI GSMYR 182 EVQLVESG 264 IIG 346 WFRQAPGKE 428 CIRVTDGSTYYTNSVKG 510RFTMSRDNAENTVYL 592 ECQRWAYPN 674 RGQGT GGLVQSGG REEVS QMNSLKPEDTAVYSRIGA QVTVSS SLRLSCAGS CAT GFSYY 183 EVQLVESG 265 IIG 347 WFRQAPGKE 429CIRVTDGSTYHTNSVKG 511 RFTMSRDNAENTVYL 593 ECQRWAYPN 675 RGQGT GGLVQSGGREEVS QMNSLKPEDTAVYS RIGA QVTVSS SLRLSCAGS CAT GFSYY 184 EVQLVESG 266YYNIG 348 WFRQAPGKE 430 CIDWTEGSTFYVDSVKG 512 RFTISTDNAKNTVYLH 594GWGRVITVQH 676 WGQGT GGLVQPGG REGVA MNSLEPEDTAVYYCAA MCADRSLFTS QVTVSSSLRLSCAAS GLTLE 185 EVQLVESG 267 YYNIG 349 WFRQAPGKE 431CIDWTDGTTFYADSVKG 513 RFTISTDNAKNTVYLH 595 GWGRVMTVQ 677 WGQGT GGLVQPGGREGVS LNSLEPEDTAVYYCAA HMCADRSLFTS QVTVSS SLRLSCAAS GLTLT 186 EVQLVESG268 SVAVG 350 WFRQAPGKE 432 WISWSGDSTYYADSVKG 514 RFTASRDNVNNTVYL 596AWKYDRASY 678 WGQGT GGLVQAGD RDFVA QMNSLKPEDTADYY DFPEAYDY QVTVSSSLRLSCAAS CAA GRTFS 187 EVQLVESG 269 SFNMG 351 WFRQTPGKE 433ATSWSDISTYYADSVKG 515 RFTISRDNAKNTVTLE 597 GYYRGGYLG 679 WGQGT GGLVQAGGREFVA MNSLKPEDTAVYYCAA YRLTLEGSYDV QVTVSS SLRLSCAAS PGTFS 188 EVQLVESG270 SFNMG 352 WFRQTPGKE 434 ATSWSDISTYYADSVKG 516 RFTISRDNAKNTVTLE 598GYYRGGYLG 680 WGQGT GGLVQAGG REFVA MNSLKPEDTAVYYCAA YRLTLEGSYDV QVTVSSSLRLSCAAS PGAFS 189 EMQLVESG 271 SFNMG 353 WFRQTPGKE 435ATSWSDISTYYADSVKG 517 RFTISRDNAKNTVTLE 599 GYYRGGYLG 681 WGQGT GGLVQAGGREFVA MNSLKPEDTAVYYCAA YRLTLEGSYDV QVTVSS SLRLSCAAS PGTFS 190 EVQLVKSG272 SFNMG 354 WFRQTPGKE 436 ATSWSDISTYYADSVKG 518 RFTISRDNAKNTVTLE 600GYYRGGYLG 682 WGQGT GGLVQAGG REFVA MNSLKPEDTAVYYCAA YRLTLEGSYDV QVTVSSSLRLSCAAS PGTFS 191 EVQLVESG 273 AMG 355 WFRQAPGKE 437 TVGWNPMNSYYGDSVKG519 RFTIFRDNAKNTVYL 601 SGSLLDVTSE 683 WGQGT GGLVQAGG REFVAQMNSLKPEDTAVYY AVYTD QVTVSS SLRLSCVVS CAG GRTFS 192 KVQLVESG 274 SYAMG356 WYRQAPGKP 438 RIYTGGTAWYEDSVKG 520 RFTISRDNAQNTVYL 602 RVRYDY 684WGQGT GGLVQAGG RELVA QMNSLKSEDTAVYY QVTVSS SLRLSCAAS CHG GSPIS 193EVQLVESG 275 SSAMG 357 WFRQAPGKD 439 AISWSGSSTHYADSVKG 521RFTISRDNAKNTMYL 603 SRRAYLPAKV 685 WGQGT GGLVQAGG RDFVA QMNSLKPADTAVYTCGEYDF QVTVSS SLRLSCAAS AA DRTFG 194 EVQLVESG 276 SYAMG 358 WFRQAPGKE 440AISLSGSMTYYADSMKG 522 RFTISRDNAKNTVYL 604 EELGDGLGYL 686 WGQGT GGLVQAGDREFVA QMNSLKPEDTAVYY AYRYDY QVTVSS SLRLSCAAS CAA GRTFS 195 EVQLVESG 277TNIMT 359 WVRQAPGKG 441 TINSGGGTTTYADSVRG 523 RFTISRDNAKNMLYL 605 PRGV687 KGRGTQ GGLVQPGG LEWIS QMSSLKPEDTALYYC VTVSS SLRLSCAAS IT GFTFS 196EVQLVESG 278 SSTMG 360 WFRQPPGKN 442 TIAWSATTTHYADAVKG 524RFTVSRDNALNTVYL 606 TLTWLGIHEY 688 WGQGT GGLVQAGG REFVA QMNSLKPEDTAVYYEYNT QVTVSS SLRLSCAAS CAA DRTFG 197 EVQLVESG 279 YYDIG 361 WFRQAPGKE 443CRFTNDGSTAYADSVKG 525 RFTISRDIVKHTVYLQ 607 GPLTKRRQCV 689 WGEGTLGGLVQPGE REGVS MNSLQPEDTAVYYC PGDFSMDF VTVSS SLRLSCTAS AA RFMLD 198EVQLVESG 280 YYDIA 362 WFRQAPGKE 444 CSFTNDGSTYYADSVKG 526RFTMSRNNDHRTVY 608 GPLTRRRQCV 690 WGEGTL GGLVQPGE REGVS LQMTSLQPEDTAVYTPGDFSMDF VTVSS SLRLSCTAS CAV RFDLD 199 EVQLVESG 281 ILTMG 363 WFRQAPGKE445 AISGIGAIHYADSVKG 527 RFTLSRDNARNTVSL 609 KANYESPSRE 691 WGQGTGGLVQAGG REFVA HMNSLKPEDTAVYYC TSYAY QVTVSS SLRLSCVAS AA GRTFS 200EVQLVESG 282 DYSMG 364 WFRQAPGKE 446 GINWSGMSTWYADSVKD 528RFTISRDNNKNTVFL 610 RQWISTIILTA 692 WGQGT GRVMQTGG LEFLA QMNSLEPGDTAVYYPSQYDY QVTVSS SLRLSCAAS CAA GHTFN 201 EVQLVESG 283 DYNMG 365 WFRQAPGKE447 GINWSGMSTWYADSVKD 529 RFTISRDNNKNTVFL 611 RQWISTIILTA 693 WGQGTGRVMQTGG LEFLA QMNSLEPGDTAVYY PSQYDY QVTVSS SLRLSCAAS CAA GHTFN 202EVQLVESG 284 EHAIG 366 WFRQAPGKE 448 LSSYLGAAYYATSVKG 530RFTISRDNAKNTVTL 612 GHFTYDDGRI 694 WGKGTL GDLVQPGG REGVS QMNSLKPEDTAVYYTIRSVDY VTVSS SLRLSCVAS CAR GFALE 203 EVQLVESG 285 EHAIG 367 WFRQAPGKE449 LSSYVGAVYYATSVKG 531 RFTISRDNAKNTVYL 613 GHFTYDDGRI 695 WGKGTLGDLVQPGG REGVS QMNSLKPEDTAVYY SIRSVDH VTVSS SLRLSCVAS CAR GFALE 204EVQLVESG 286 TAVMG 368 WFRQAPGKE 450 MISWSGSMTYYAKSVKG 532RFTISRDNAKNTMYL 614 DMGGGPPDG 696 WGKGTL GGLVQAGG RGLVA QMNSLKPEDTAVYYDAMPRLSSG VTVSS SLRLSCVAS CAA MDY GRTLS 205 EVQLVESG 287 RFSIDA 369WYRQAPGKQ 451 TVTEDGTKNYADSVKG 533 RATISRDDANNSMYL 615 GGLIDGAAPY 697WGRGT GGLVQAGG MG RELVA EMNTLKPEDTAVYYC EF QVTVSS SLRLSCAAS KM GNDFA 206EMQLVESG 288 AGAMG 370 WYRQPAGKQ 452 DITLGGSTNYADSVKG 534RFTISRDNAKNAVFL 616 LINTFARKIPR 698 WGQGT GGWVQAG RELVA QMNSLKPEDTAVYYYA QVTVSS GSLRLSCAS CNG SGSIFS 207 EVQLVESG 289 GRYTMG 371 WFRQAPGRE 453AISWIGGRTYYVDVVKG 535 RFTISRDNAKKMVYL 617 AFQALGSPRE 699 WGQGT GGLVQAGGREFVA QMNSLKPDDTAVYH YDY QVTVSS SLRLSCAAS CAA GPTTF

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1;or from the group of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” (as defined herein) with at least one ofthe CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the Nanobodies of theinvention bind to ion channels such as e.g. P2X7 with an affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table B-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table B-1; and/or from thegroup consisting of the CDR3 sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the CDR3 sequences listed inTable B-1.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” with at least one of the CDR1, CDR2 andCDR3 sequences, respectively, listed in Table B-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table B-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table B-1, respectively; andat least one of the CDR1 and CDR2 sequences present is suitably chosenfrom the group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table B-1 or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table B-1; and/or from the group consisting of the CDR1 andCDR2 sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table B-1.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1 or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, at east one ofthe CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table B-1. Preferably, in this aspect, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the corresponding CDR sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR sequences that have 3, 2 or only 1 amino acid difference(s) withat least one of the corresponding sequences, respectively, listed inTable B-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table B-1. Preferably, in this aspect, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with the CDR1and CDR2 sequences, respectively, listed in Table B-1; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table B-1. Preferably, in this aspect, the remaining CDRsequence present is suitably chosen from the group of CDR sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with at leastone of the corresponding CDR sequences listed in Table B-1; and/or fromthe group consisting of CDR sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the corresponding sequenceslisted in Table B-1

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table B-1, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table B-1. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable B-1; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the corresponding CDRsequences listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1. Also, generally, the combinations of CDR's listed in TableB-1 (i.e. those mentioned on the same line in Table B-1) are preferred.Thus, it is generally preferred that, when a CDR in a Nanobody of theinvention is a CDR sequence mentioned in Table B-1 or is suitably chosenfrom the group of CDR sequences that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity with a CDR sequence listed in Table B-1; and/orfrom the group consisting of CDR sequences that have 3, 2 or only 1amino acid difference(s) with a CDR sequence listed in Table B-1 that atleast one and preferably both of the other CDR's are suitably chosenfrom the CDR sequences that belong to the same combination in Table B-1(i.e. mentioned on the same line in Table B-1) or are suitably chosenfrom the group of CDR sequences that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity with the CDR sequence(s) belonging to the samecombination and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the CDR sequence(s)belonging to the same combination. The other preferences indicated inthe above paragraphs also apply to the combinations of CDR's mentionedin Table B-1.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table B-1, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table B-1 (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table B-1; a CDR2 sequence that has 3, 2 or1 amino acid difference with one of the CDR2 sequences mentioned inTable B-1 (but belonging to a different combination); and a CDR3sequence that has more than 80% sequence identity with one of the CDR3sequences mentioned in Table B-1 (but belonging to a differentcombination); or (2) a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table B-1; a CDR2sequence, and one of the CDR3 sequences listed in Table B-1; or (3) aCDR1 sequence; a CDR2 sequence that has more than 80% sequence identitywith one of the CDR2 sequence listed in Table B-1; and a CDR3 sequencethat has 3, 2 or 1 amino acid differences with the CDR3 sequencementioned in Table B-1 that belongs to the same combination as the CDR2sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table B-1; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table B-1 that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table B-1 that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table B-1 and a CDR3 sequence listed inTable B-1 (in which the CDR2 sequence and CDR3 sequence may belong todifferent combinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table B-1; the CDR2 sequencelisted in Table B-1 that belongs to the same combination; and a CDR3sequence mentioned in Table B-1 that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table B-1; a CDR2 sequence that has3, 2 or 1 amino acid differences with the CDR2 sequence mentioned inTable B-1 that belongs to the same combination; and a CDR3 sequence thathas more than 80% sequence identity with the CDR3 sequence listed inTable B-1 that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table B-1, a CDR2 sequence thathas more than 80% sequence identity with the CDR2 sequence mentioned inTable B-1 that belongs to the same combination; and the CDR3 sequencementioned in Table B-1 that belongs to the same combination. In the mostpreferred Nanobodies of the invention, the CDR1, CDR2 and CDR3 sequencespresent are suitably chosen from one of the combinations of CDR1, CDR2and CDR3 sequences, respectively, listed in Table B-1.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences (as defined herein) have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with the CDR sequencesof at least one of the immunoglobulin sequences of SEQ ID NO's: 705 to788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772to 773, 775, or 778 to 780, more preferably SEQ ID NO's 726 to 750, 753to 758, 762 to 764, 772 to 773, 775, or 778 to 780, more preferred SEQID NO's 732, 773 or 778 (see Table A-1).

Generally, Nanobodies with the above CDR sequences may be as furtherdescribed herein, and preferably have framework sequences that are alsoas further described herein. Thus, for example and as mentioned herein,such Nanobodies may be naturally occurring Nanobodies (from any suitablespecies), naturally occurring V_(HH), sequences (i.e. from a suitablespecies of Camelid) or synthetic or semi-synthetic immunoglobulinsequences or Nanobodies, including but not limited to partiallyhumanized Nanobodies or V_(HH) sequences, fully humanized Nanobodies orV_(HH) sequences, camelized heavy chain variable domain sequences, aswell as Nanobodies that have been obtained by the techniques mentionedherein.

Thus, in one specific, but non-limiting aspect, the invention relates toa humanized Nanobody, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 to CDR3 are as defined herein and in whichsaid humanized Nanobody comprises at least one humanizing substitution(as defined herein), and in particular at least one humanizingsubstitution in at least one of its framework sequences (as definedherein).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the immunoglobulin sequences of SEQ ID NO's: 705 to788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772to 773, 775, or 778 to 780, more preferably SEQ ID NO's 726 to 750, 753to 758, 762 to 764, 772 to 773, 775, or 778 to 780, more preferred SEQID NO's 732, 773 or 778 (see Table A-1). This degree of amino acididentity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said Nanobodyand one or more of the sequences of SEQ ID NO's: 705 to 788, morepreferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773,775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or 778 (seeTable A-1), in which the amino acid residues that form the frameworkregions are disregarded. Such Nanobodies can be as further describedherein.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody with an immunoglobulin sequence that is chosen from the groupconsisting of SEQ ID NO's: 705 to 788, more preferably SEQ ID NO's 726to 750, 753 to 758, 762 to 764, 772 to 773, 775, or 778 to 780, morepreferred SEQ ID NO's 732, 773 or 778 (see Table A-1) or from the groupconsisting of from immunoglobulin sequences that have more than 80%,preferably more than 90%, more preferably more than 95%, such as 99% ormore sequence identity (as defined herein) with at least one of theimmunoglobulin sequences of SEQ ID NO's: 705 to 788, more preferably SEQID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or 778 to780, more preferred SEQ ID NO's 732, 773 or 778 (see Table A-1).

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the Nanobodies of SEQ ID NO's: 705 to 788, morepreferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773,775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or 778 (seeTable A-1), that comprise, compared to the corresponding native V_(HH)sequence, at least one humanizing substitution (as defined herein), andin particular at least one humanizing substitution in at least one ofits framework sequences (as defined herein).

The polypeptides of the invention comprise or essentially consist of atleast one Nanobody of the invention. Some preferred, but non-limitingexamples of polypeptides of the invention are given in SEQ ID NO's: 789to 791 (see Table A-3).

TABLE A-3 (if applicable): Preferred polypeptide or compound sequences(also referred herein as a sequence with a particular name or SEQ ID NO:X, wherein X is a number referring to the relevant immunoglobulinsequence): P2X7 SEQ ID NO: X, Name function wherein X = Immunoglobulinsequence 14D5- Enhancer 789 EVQLVESGGGLVQAGGSLRLSCAASGSPISSY 35GS- (seeAMGWYRQAPGKPRELVARIYTGGTAWYEDSV 14D5 exampleKGRFTISRDNAQNTVYLQMNSLKSEDTAVYYC 3.11&3.12)HGRVRYDYWGQGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSPISSYAMG WYRQAPGKPRELVARIYTGGTAWYEDSVKGRFTISRDNAQNTVYLQMNSLKSEDTAVYYCHGR VRYDYWGQGTQVTVSS 13A7- Blocker 790EVQLVESGGGLVQPGESLRLSCTASRFMLDYY 35GS- (seeDIGWFRQAPGKEREGVSCRFTNDGSTAYADS 13A7 exampleVKGRFTISRDIVKHTVYLQMNSLQPEDTAVYYC 3.11&3.12)AAGPLTKRRQCVPGDFSMDFWGEGTLVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGESLRLSCTA SRFMLDYYDIGWFRQAPGKEREGVSCRFTNDGSTAYADSVKGRFTISRDIVKHTVYLQMNSLQP EDTAVYYCAAGPLTKRRQCVPGDFSMDFWGEGTLVTVSS 8G11- Blocker 791 EVQLVESGGGLVQAGGSLRLSCAASGNFFRV 35GS- (seeNTMAWYRQAPGKQRELVADITRGDRTNYADT 8G11 exampleVNGRFTISRDNVRNTVYLQMNGLRPEDTAAYY 3.11&3.12)CYAVIELGVLEPRDYWGQGTQVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGNFF RVNTMAWYRQAPGKQRELVADITRGDRTNYADTVNGRFTISRDNVRNTVYLQMNGLRPEDTAA YYCYAVIELGVLEPRDYWGQGTQVTVSS

It will be clear to the skilled person that the Nanobodies that arementioned herein as “preferred” (or “more preferred”, “even morepreferred”, etc.) are also preferred (or more preferred, or even morepreferred, etc.) for use in the polypeptides described herein. Thus,polypeptides that comprise or essentially consist of one or more“preferred” Nanobodies of the invention will generally be preferred, andpolypeptides that comprise or essentially consist of one or more “morepreferred” Nanobodies of the invention will generally be more preferred.

Generally, proteins or polypeptides that comprise or essentially consistof a single Nanobody (such as a single Nanobody of the invention) willbe referred to herein as “monovalent” proteins or polypeptides or as“monovalent constructs”. Proteins and polypeptides that comprise oressentially consist of two or more Nanobodies (such as at least twoNanobodies of the invention or at least one Nanobody of the inventionand at least one other Nanobody) will be referred to herein as“multivalent” proteins or polypeptides or as “multivalent constructs”,and these may provide certain advantages compared to the correspondingmonovalent Nanobodies of the invention. Some non-limiting examples ofsuch multivalent constructs will become clear from the furtherdescription herein.

According to one specific, but non-limiting aspect, a polypeptide of theinvention comprises or essentially consists of at least two Nanobodiesof the invention, such as two or three Nanobodies of the invention. Asfurther described herein, such multivalent constructs can providecertain advantages compared to a protein or polypeptide comprising oressentially consisting of a single Nanobody of the invention, such as amuch improved avidity for ion channels such as e.g. P2X7. Suchmultivalent constructs will be clear to the skilled person based on thedisclosure herein; some preferred, but non-limiting examples of suchmultivalent Nanobody constructs are the constructs of SEQ ID NO's: 789to 791.

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least one Nanobodyof the invention and at least one other binding unit (i.e. directedagainst another epitope, antigen, target, protein or polypeptide), whichis preferably also a Nanobody. Such proteins or polypeptides are alsoreferred to herein as “multispecific” proteins or polypeptides or as‘multispecific constructs”, and these may provide certain advantagescompared to the corresponding monovalent Nanobodies of the invention (aswill become clear from the further discussion herein of some preferred,but-nonlimiting multispecific constructs). Such multispecific constructswill be clear to the skilled person based on the disclosure herein; somepreferred, but non-limiting examples of such multispecific Nanobodyconstructs are the constructs of SEQ ID NO's: 789 to 791.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one Nanobody of the invention, optionally one or more furtherNanobodies, and at least one other immunoglobulin sequence (such as aprotein or polypeptide) that confers at least one desired property tothe Nanobody of the invention and/or to the resulting fusion protein.Again, such fusion proteins may provide certain advantages compared tothe corresponding monovalent Nanobodies of the invention. Somenon-limiting examples of such immunoglobulin sequences and of suchfusion constructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, forexample to provide a trivalent bispecific construct comprising twoNanobodies of the invention and one other Nanobody, and optionally oneor more other immunoglobulin sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more Nanobodies and/or otherimmunoglobulin sequences may be directly linked to each other and/orsuitably linked to each other via one or more linker sequences. Somesuitable but non-limiting examples of such linkers will become clearfrom the further description herein.

In one specific aspect of the invention, a Nanobody of the invention ora compound, construct or polypeptide of the invention comprising atleast one Nanobody of the invention may have an increased half-life,compared to the corresponding immunoglobulin sequence of the invention.Some preferred, but non-limiting examples of such Nanobodies, compoundsand polypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise u Nanobodiessequences or polypeptides of the invention that have been chemicallymodified to increase the half-life thereof (for example, by means ofpegylation); immunoglobulin sequences of the invention that comprise atleast one additional binding site for binding to a serum protein (suchas serum albumin, see for example EP 0 368 684 B1, page 4); orpolypeptides of the invention that comprise at least one Nanobody of theinvention that is linked to at least one moiety (and in particular atleast one immunoglobulin sequence) that increases the half-life of theNanobody of the invention. Examples of polypeptides of the inventionthat comprise such half-life extending moieties or immunoglobulinsequences will become clear to the skilled person based on the furtherdisclosure herein; and for example include, without limitation,polypeptides in which the one or more Nanobodies of the invention aresuitable linked to one or more serum proteins or fragments thereof (suchas serum albumin or suitable fragments thereof) or to one or morebinding units that can bind to serum proteins (such as, for example,Nanobodies or (single) domain antibodies that can bind to serum proteinssuch as serum albumin, serum immunoglobulins such as IgG, ortransferrin); polypeptides in which a Nanobody of the invention islinked to an Fc portion (such as a human Fc) or a suitable part orfragment thereof; or polypeptides in which the one or more Nanobodies ofthe invention are suitable linked to one or more small proteins orpeptides that can bind to serum proteins (such as, without limitation,the proteins and peptides described in WO 91/01743, WO 01/45746, WO02/076489 and to the US provisional application of Ablynx N.V. entitled“Peptides capable of binding to serum proteins” of Ablynx N.V. filed onDec. 5, 2006 (see also PCT/EP/2007/063348).

Again, as will be clear to the skilled person, such Nanobodies,compounds, constructs or polypeptides may contain one or more additionalgroups, residues, moieties or binding units, such as one or more furtherimmunoglobulin sequences and in particular one or more additionalNanobodies (i.e. not directed against ion channels such as e.g. P2X7),so as to provide a tri- of multispecific Nanobody construct.

Generally, the Nanobodies of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the correspondingimmunoglobulin sequence of the invention per se. For example, theNanobodies, compounds, constructs or polypeptides of the invention withincreased half-life may have a half-life that is increased with morethan 1 hours, preferably more than 2 hours, more preferably more than 6hours, such as more than 12 hours, or even more than 24, 48 or 72 hours,compared to the corresponding immunoglobulin sequence of the inventionper se.

In a preferred, but non-limiting aspect of the invention, suchNanobodies, compound, constructs or polypeptides of the inventionexhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), preferably at least 9 days (such as about9 to 14 days), more preferably at least about 10 days (such as about 10to 15 days), or at least about 11 days (such as about 11 to 16 days),more preferably at least about 12 days (such as about 12 to 18 days ormore), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) Nanobodies of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) immunoglobulin sequencesthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more immunoglobulin sequencesthat allow the resulting polypeptides of the invention to cross theblood brain barrier may be one or more (such as two and preferably one)Nanobodies, such as the Nanobodies described in WO 02/057445, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In particular, polypeptides comprising one or more Nanobodies of theinvention are preferably such that they:

-   -   bind to ion channels such as e.g. P2X7 with a dissociation        constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and        preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably        10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association constant        (K_(A)) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷        to 10¹² liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that they:    -   bind to ion channels such as e.g. P2X7 with a k_(on)-rate of        between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³        M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and        10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to ion channels such as e.g. P2X7 with a k_(off) rate        between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near        irreversible complex with a t_(1/2) of multiple days),        preferably between 10⁻² s⁻¹ and 10⁻⁵ s⁻¹, more preferably        between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s¹ and 10⁻⁶        s⁻¹.

Preferably, a polypeptide that contains only one immunoglobulin sequenceof the invention is preferably such that it will bind to ion channelssuch as e.g. P2X7 with an affinity less than 500 nM, preferably lessthan 200 nM, more preferably less than 10 nM, such as less than 500 μM.In this respect, it will be clear to the skilled person that apolypeptide that contains two or more Nanobodies of the invention maybind to ion channels such as e.g. P2X7 with an increased avidity,compared to a polypeptide that contains only one immunoglobulin sequenceof the invention.

Some preferred IC₅₀ values for binding of the immunoglobulin sequencesor polypeptides of the invention to ion channels such as e.g. P2X7 willbecome clear from the further description and examples herein.

Other polypeptides according to this preferred aspect of the inventionmay for example be chosen from the group consisting of immunoglobulinsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the immunoglobulin sequences of SEQID NO's: 789 to 791 (see Table A-3), in which the Nanobodies comprisedwithin said immunoglobulin sequences are preferably as further definedherein.

Another aspect of this invention relates to a nucleic acid that encodesan immunoglobulin sequence of the invention (such as a Nanobody of theinvention) or a polypeptide of the invention comprising the same. Again,as generally described herein for the nucleic acids of the invention,such a nucleic acid may be in the form of a genetic construct, asdefined herein.

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing an immunoglobulin sequence(such as a Nanobody) of the invention and/or a polypeptide of theinvention comprising the same; and/or that contains a nucleic acid ofthe invention. Some preferred but non-limiting examples of such hosts orhost cells will become clear from the further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one immunoglobulin sequence of theinvention, at least one polypeptide of the invention and/or at least onenucleic acid of the invention, and optionally one or more furthercomponents of such compositions known per se, i.e. depending on theintended use of the composition. Such a product or composition may forexample be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention further relates to methods for preparing or generating theimmunoglobulin sequences, compounds, constructs, polypeptides, nucleicacids, host cells, products and compositions described herein. Somepreferred but non-limiting examples of such methods will become clearfrom the further description herein.

The invention further relates to applications and uses of theimmunoglobulin sequences, compounds, constructs, polypeptides, nucleicacids, host cells, products and compositions described herein, as wellas to methods for the prevention and/or treatment for diseases anddisorders associated with ion channels such as e.g. P2X7. Some preferredbut non-limiting applications and uses will become clear from thefurther description herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description hereinbelow.Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can generally beobtained by any of the techniques (1) to (8) mentioned on pages 61 and62 of WO 08/020079, or any other suitable technique known per se. Onepreferred class of Nanobodies corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed against ion channelssuch as e.g. P2X7. As further described herein, such V_(HH) sequencescan generally be generated or obtained by suitably immunizing a speciesof Camelid with ion channels such as e.g. P2X7 (i.e. so as to raise animmune response and/or heavy chain antibodies directed against ionchannels such as e.g. P2X7), by obtaining a suitable biological samplefrom said Camelid (such as a blood sample, serum sample or sample ofB-cells), and by generating V_(HH) sequences directed against ionchannels such as e.g. P2X7, starting from said sample, using anysuitable technique known per se. Such techniques will be clear to theskilled person and/or are further described herein.

Alternatively, such naturally occurring V_(HH) domains against ionchannels such as e.g. P2X7, can be obtained from naïve libraries ofCamelid V_(HH) sequences, for example by screening such a library usingion channels such as e.g. P2X7, or at least one part, fragment,antigenic determinant or epitope thereof using one or more screeningtechniques known per se. Such libraries and techniques are for exampledescribed in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694.Alternatively, improved synthetic or semi-synthetic libraries derivedfrom naïve V_(HH) libraries may be used, such as V_(HH) librariesobtained from naïve V_(HH) libraries by techniques such as randommutagenesis and/or CDR shuffling, as for example described in WO00/43507.

Thus, in another aspect, the invention relates to a method forgenerating Nanobodies, that are directed against ion channels such ase.g. P2X7. In one aspect, said method at least comprises the steps of:

-   a) providing a set, collection or library of Nanobody sequences; and-   b) screening said set, collection or library of Nanobody sequences    for Nanobody sequences that can bind to and/or have affinity for ion    channels such as e.g. P2X7;    and-   c) isolating the Nanobody or Nanobodies that can bind to and/or have    affinity for ion channels such as e.g. P2X7.

In such a method, the set, collection or library of Nanobody sequencesmay be a naïve set, collection or library of Nanobody sequences; asynthetic or semi-synthetic set, collection or library of Nanobodysequences; and/or a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofNanobody sequences may be an immune set, collection or library ofNanobody sequences, and in particular an immune set, collection orlibrary of V_(HH) sequences, that have been derived from a species ofCamelid that has been suitably immunized with ion channels such as e.g.P2X7 or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

In the above methods, the set, collection or library of Nanobody orV_(HH) sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) Nanobodysequences will be clear to the person skilled in the art, for example onthe basis of the further disclosure herein. Reference is also made to WO03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23,9, 1105-1116 (2005).

In another aspect, the method for generating Nanobody sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species    of Camelid that express immunoglobulin sequences;-   b) screening said collection or sample of cells for (i) cells that    express an immunoglobulin sequence that can bind to and/or have    affinity for ion channels such as e.g. P2X7; and (ii) cells that    express heavy chain antibodies, in which substeps (i) and (ii) can    be performed essentially as a single screening step or in any    suitable order as two separate screening steps, so as to provide at    least one cell that expresses a heavy chain antibody that can bind    to and/or has affinity for ion channels such as e.g. P2X7;    and-   c) either (i) isolating from said cell the V_(HH) sequence present    in said heavy chain antibody; or (ii) isolating from said cell a    nucleic acid sequence that encodes the V_(HH) sequence present n    said heavy chain antibody, followed by expressing said V_(HH)    domain.

In the method according to this aspect, the collection or sample ofcells may for example be a collection or sample of B-cells. Also, inthis method, the sample of cells may be derived from a Camelid that hasbeen suitably immunized with ion channels such as e.g. P2X7 or asuitable antigenic determinant based thereon or derived therefrom, suchas an antigenic part, fragment, region, domain, loop or other epitopethereof. In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820. Particular reference is made to the so-called“Nanoclone™” technique described in International application WO06/079372 by Ablynx N.V.

In another aspect, the method for generating an immunoglobulin sequencedirected against ion channels such as e.g. P2X7 may comprise at leastthe steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding heavy chain antibodies or Nanobody sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode a heavy chain    antibody or a Nanobody sequence that can bind to and/or has affinity    for ion channels such as e.g. P2X7;    and-   c) isolating said nucleic acid sequence, followed by expressing the    V_(HH) sequence present in said heavy chain antibody or by    expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or Nanobody sequences may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of heavy chain antibodies orV_(HH) sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofNanobody sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or V_(HH)sequences derived from a Camelid that has been suitably immunized withion channels such as e.g. P2X7 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding immunoglobulin sequences will be clear to the person skilled inthe art, for example on the basis of the further disclosure herein.Reference is also made to WO 03/054016 and to the review by Hoogenboomin Nature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of themethods described herein can also be performed as a selection step.Accordingly the term “screening” as used in the present description cancomprise selection, screening or any suitable combination of selectionand/or screening techniques. Also, when a set, collection or library ofsequences is used, it may contain any suitable number of sequences, suchas 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collectionor library of immunoglobulin sequences may be obtained or defined byrational, or semi-empirical approaches such as computer modellingtechniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library)), or any other source of diverse sequences (asdescribed for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection orlibrary of sequences can be displayed on the surface of a phageparticle, a ribosome, a bacterium, a yeast cell, a mammalian cell, andlinked to the nucleotide sequence encoding the immunoglobulin sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired immunoglobulinsequences of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences or Nanobodysequences directed against ion channels such as e.g. P2X7 involvessuitably immunizing a transgenic mammal that is capable of expressingheavy chain antibodies (i.e. so as to raise an immune response and/orheavy chain antibodies directed against ion channels such as e.g. P2X7),obtaining a suitable biological sample from said transgenic mammal thatcontains (nucleic acid sequences encoding) said V_(HH) sequences orNanobody sequences (such as a blood sample, serum sample or sample ofB-cells), and then generating V_(HH) sequences directed against ionchannels such as e.g. P2X7, starting from said sample, using anysuitable technique known per se (such as any of the methods describedherein or a hybridoma technique). For example, for this purpose, theheavy chain antibody-expressing mice and the further methods andtechniques described in WO 02/085945, WO 04/049794 and WO 06/008548 andJanssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10;103(41):15130-5 can be used. For example, such heavy chain antibodyexpressing mice can express heavy chain antibodies with any suitable(single) variable domain, such as (single) variable domains from naturalsources (e.g. human (single) variable domains, Camelid (single) variabledomains or shark (single) variable domains), as well as for examplesynthetic or semi-synthetic (single) variable domains.

The invention also relates to the V_(HH) sequences or Nanobody sequencesthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or immunoglobulin sequenceof said V_(HH) sequence or Nanobody sequence; and of expressing orsynthesizing said V_(HH) sequence or Nanobody sequence in a manner knownper se, such as by expression in a suitable host cell or host organismor by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of theinvention comprises Nanobodies with an immunoglobulin sequence thatcorresponds to the immunoglobulin sequence of a naturally occurringV_(HH) domain, but that has been “humanized”, i.e. by replacing one ormore amino acid residues in the immunoglobulin sequence of saidnaturally occurring V_(HH) sequence (and in particular in the frameworksequences) by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above), as further describedon, and using the techniques mentioned on, page 63 of WO 08/020079.Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an immunoglobulin sequence that corresponds tothe immunoglobulin sequence of a naturally occurring V_(H) domain, butthat has been “camelized”, i.e. by replacing one or more amino acidresidues in the immunoglobulin sequence of a naturally occurring V_(H)domain from a conventional 4-chain antibody by one or more of the aminoacid residues that occur at the corresponding position(s) in a V_(HH)domain of a heavy chain antibody, as further described on, and using thetechniques mentioned on, page 63 of WO 08/020079.

Other suitable methods and techniques for obtaining the Nanobodies ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably V_(HH) sequences, willbe clear from the skilled person, and may for example include thetechniques that are mentioned on page 64 of WO 08/00279 As mentionedherein, Nanobodies may in particular be characterized by the presence ofone or more “Hallmark residues” (as described herein) in one or more ofthe framework sequences.

Thus, according to one preferred, but non-limiting aspect of theinvention, a Nanobody in its broadest sense can be generally defined asa polypeptide comprising:

-   a) an immunoglobulin sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an immunoglobulin sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 45    according to the Kabat numbering is a charged amino acid (as defined    herein) or a cysteine residue, and position 44 is preferably an E;    and/or:-   c) an immunoglobulin sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q; and/or in which:

-   b) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid or a cysteine and the amino acid    residue at position 44 according to the Kabat numbering is    preferably E;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody in its broadest sense can be generally definedas a polypeptide comprising:

-   a) an immunoglobulin sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an immunoglobulin sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an immunoglobulin sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody ofthe invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and 5, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody against ion channels such as e.g. P2X7according to the invention may have the structure:

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, according to one preferred, but non-limiting aspect ofthe invention, a Nanobody can generally be defined as a polypeptidecomprising an immunoglobulin sequence that is comprised of fourframework regions/sequences interrupted by three complementaritydetermining regions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q; or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R;    -   and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q;    and in which:

-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:

-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;    and in which

-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:

-   d) CDR1. CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:

-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R;    and in which:

-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:

-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:

-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:

-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:

-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to (a-1) to(a-4) above; according to b) above; according to (b-1) to (b-4) above;according to (c) above; and/or according to (c-1) to (c-4) above, inwhich either:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as    described herein) and the amino acid residue at position 108 is Q;    or in which:-   ii) the amino acid residues at positions 43-46 according to the    Kabat numbering form the sequence KERE or KQRE (or a KERE-like    sequence as described) and the amino acid residue at position 108 is    Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively; and in        which:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KEREor KQRE, the amino acid residue at position 37 is most preferably F. Inthe Nanobodies of the invention in which the amino acid residues atpositions 44-47 according to the Kabat numbering form the sequence GLEW,the amino acid residue at position 37 is chosen from the groupconsisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified on the basis of thefollowing three groups:

-   i) The “GLEW-group”: Nanobodies with the immunoglobulin sequence    GLEW at positions 44-47 according to the Kabat numbering and Q at    position 108 according to the Kabat numbering. As further described    herein, Nanobodies within this group usually have a V at position    37, and can have a W, P, R or S at position 103, and preferably have    a W at position 103. The GLEW group also comprises some GLEW-like    sequences such as those mentioned in Table B-2 below. More    generally, and without limitation, Nanobodies belonging to the    GLEW-group can be defined as Nanobodies with a G at position 44    and/or with a W at position 47, in which position 46 is usually E    and in which preferably position 45 is not a charged amino acid    residue and not cysteine;-   ii) The “KERE-group”: Nanobodies with the immunoglobulin sequence    KERE or KQRE (or another KERE-like sequence) at positions 43-46    according to the Kabat numbering and Q or L at position 108    according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a F at position 37, an L    or F at position 47; and can have a W, P, R or S at position 103,    and preferably have a W at position 103. More generally, and without    limitation, Nanobodies belonging to the KERE-group can be defined as    Nanobodies with a K, Q or R at position 44 (usually K) in which    position 45 is a charged amino acid residue or cysteine, and    position 47 is as further defined herein;-   iii) The “103 P, R, S-group”: Nanobodies with a P, R or S at    position 103. These Nanobodies can have either the immunoglobulin    sequence GLEW at positions 44-47 according to the Kabat numbering or    the immunoglobulin sequence KERE or KQRE at positions 43-46    according to the Kabat numbering, the latter most preferably in    combination with an F at position 37 and an L or an F at position 47    (as defined for the KERE-group); and can have Q or L at position 108    according to the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. havecharacteristics of) two or more of these classes. For example, onespecifically preferred group of Nanobodies has GLEW or a GLEW-likesequence at positions 44-47; P, R or S (and in particular R) at position103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred toabove describe and apply to Nanobodies in the form of a native (i.e.non-humanized) V_(HH) sequence, and that humanized variants of theseNanobodies may contain other amino acid residues than those indicatedabove (i.e. one or more humanizing substitutions as defined herein). Forexample, and without limitation, in some humanized Nanobodies of theGLEW-group or the 103 P, R, S-group, Q at position 108 may be humanizedto 108L. As already mentioned herein, other humanizing substitutions(and suitable combinations thereof) will become clear to the skilledperson based on the disclosure herein. In addition, or alternatively,other potentially useful humanizing substitutions can be ascertained bycomparing the sequence of the framework regions of a naturally occurringV_(HH) sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized V_(HH) sequences can be tested for affinity for the target,for stability, for ease and level of expression, and/or for otherdesired properties. In this way, by means of a limited degree of trialand error, other suitable humanizing substitutions (or suitablecombinations thereof) can be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) a Nanobody may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, ft S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferred aspectsherein, and are more preferably as defined according to one of the morepreferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P, R,S residues mentioned above, the Nanobodies of the invention can contain,at one or more positions that in a conventional V_(H) domain would form(part of) the V_(H)/V_(L) interface, one or more amino acid residuesthat are more highly charged than the amino acid residues that naturallyoccur at the same position(s) in the corresponding naturally occurringV_(H) sequence, and in particular one or more charged amino acidresidues (as mentioned in Table A-2 on page 48 of the Internationalapplication WO 08/020079). Such substitutions include, but are notlimited to, the GLEW-like sequences mentioned in Table B-2 below; aswell as the substitutions that are described in the InternationalApplication WO 00/29004 for so-called “microbodies”, e.g. so as toobtain a Nanobody with Q at position 108 in combination with KLEW atpositions 44-47. Other possible substitutions at these positions will beclear to the skilled person based upon the disclosure herein. In oneaspect of the Nanobodies of the invention, the amino acid residue atposition 83 is chosen from the group consisting of L, M, S, V and W; andis preferably L. Also, in one aspect of the Nanobodies of the invention,the amino acid residue at position 83 is chosen from the groupconsisting of R, K, N, E, G, I, T and Q; and is most preferably either Kor E (for Nanobodies corresponding to naturally occurring V_(HH)domains) or R (for “humanized” Nanobodies, as described herein). Theamino acid residue at position 84 is chosen from the group consisting ofP, A, R, S, D T, and V in one aspect, and is most preferably P (forNanobodies corresponding to naturally occurring V_(HH) domains) or R(for “humanized” Nanobodies, as described herein). Furthermore, in oneaspect of the Nanobodies of the invention, the amino acid residue atposition 104 is chosen from the group consisting of G and D; and is mostpreferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human V_(H) domain, V_(H)3, aresummarized in Table B-2.

Some especially preferred but non-limiting combinations of theseHallmark Residues as occur in naturally occurring V_(E) domains arementioned in Table B-3. For comparison, the corresponding amino acidresidues of the human V_(H)3 called DP-47 have been indicated initalics.

TABLE B-2 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues  11 L, V; L, S, V, M, W, F, T, Q, E, A, R, G, K, Y,predominantly L N, P, I; preferably L  37 V, I, F; F⁽¹⁾, Y, V, L, A, H,S, I, W, C, N, G, D, usually V T, P, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G E⁽³⁾,Q⁽³⁾, G⁽²⁾, D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G⁽²⁾,E⁽³⁾ or Q⁽³⁾; most preferably G⁽²⁾ or Q⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, P, H,F, G, Q, S, E, T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, YF⁽¹⁾, L⁽¹⁾ or W⁽²⁾ G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D;preferably W⁽²⁾, L⁽¹⁾ or F⁽¹⁾  83 R or K; R, K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I,V, G, M, L, A, usually R D, Y, H; preferably K or R; most preferably K 84 A, T, D; P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R, Y, N, Q, predominantlyA G, E; preferably P 103 W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V,Q, P⁽⁶⁾, E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L;preferably G 108 L, M or T; Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H;predominantly L preferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but notexclusively, in combination with KERE or KQRE at positions 43-46.⁽²⁾Usually as GLEW at positions 44-47. ⁽³⁾Usually as KERE or KQRE atpositions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF, KEREG, KQREW orKQREG at positions 43-47. Alternatively, also sequences such as TERE(for example TEREL), TQRE (for example TQREL), KECE (for example KECELor KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE (forexample RQREL, RQREF or RQREW), QERE (for example QEREG), QQRE, (forexample QQREW, QQREL or QQREF), KGRE (for example KGREG), KDRE (forexample KDREV) are possible. Some other possible, but less preferredsequences include for example DECKL and NVCEL. ⁽⁴⁾With both GLEW atpositions 44-47 and KERE or KQRE at positions 43-46. ⁽⁵⁾Often as KP orEP at positions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾Inparticular, but not exclusively, in combination with GLEW at positions44-47. ⁽⁷⁾With the proviso that when positions 44-47 are GLEW, position108 is always Q in (non-humanized) V_(HH) sequences that also contain aW at 103. ⁽⁸⁾The GLEW group also contains GLEW-like sequences atpositions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW,ELEW, GPEW, EWLP, GPER, GLER and ELEW.

TABLE B-3 Some preferred but non-limiting combinations of HallmarkResidues in naturally occurring Nanobodies. For humanization of thesecombinations, reference is made to the specification. 11 37 44 45 47 8384 103 104 108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R LK P W G Q L F E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q LF L R V K P Q G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group LV G L W K S W G Q M V G L W K P R G Q

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH), domain.

Such amino acid residues will be clear to the skilled person. Tables B-4to B-7 mention some non-limiting residues that can be present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of naturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering by Chothia (supra) that the residues at thesepositions already form part of CDR1).

In Tables B-4-B-7, some of the non-limiting residues that can be presentat each position of a human V_(H)3 domain have also been mentioned.Again, for each position, the amino acid residue that most frequentlyoccurs at each position of a naturally occurring human V_(H)3 domain isindicated in bold; and other preferred amino acid residues have beenunderlined.

For reference only, Tables B-4-B-7 also contain data on the V_(HH)entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at eachamino acid position for a representative sample of 7732 V_(HH) sequences(including a.o. data kindly provided by David Lutje Hulsing and Prof.Theo Verrips of Utrecht University). The values for the V_(HH) entropyand the V_(HH) variability provide a measure for the variability anddegree of conservation of amino acid residues between the 7732 V_(HH)sequences analyzed: low values (i.e. <1, such as <0.5) indicate that anamino acid residue is highly conserved between the V_(HH) sequences(i.e. little variability). For example, the G at position 9 and the W atposition 36 have values for the V_(HH) entropy of 0.01 and 0respectively, indicating that these residues are highly conserved andhave little variability (and in case of position 36 is W in all 7732sequences analysed), whereas for residues that form part of the CDR'sgenerally values of 1.5 or more are found (data not shown). Note thatthe data represented below support the hypothesis that the amino acidresidues at positions 27-30 and maybe even also at positions 93 and 94already form part of the CDR's (although the invention is not limited toany specific hypothesis or explanation, and as mentioned above, hereinthe numbering according to Kabat is used). For a general explanation ofsequence entropy, sequence variability and the methodology fordetermining the same, see Oliveira et al., PROTEINS: Structure, Functionand Genetics, 52: 544-552 (2003).

TABLE B-4 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table B-2) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 1 E, Q E, Q, K, D,A, G, R 0.47 5 2 V V, M, A, E, L 0.04 1 3 Q Q, K, P, H, F, R 0.04 1 4 LL, M, Q, P, R, F, V 0.02 1 5 V, L V, Q, M, E, A, L, P, K, R 0.35 3 6 EE, A, Q, D, K, H 0.21 5 7 S, T S, F, L, W, T 0.05 2 8 G, R G, R, E, V0.04 1 9 G G, R, V, A 0.01 1 10 G, V G, D, R, S, K, E, A, Q, N, T, V0.22 4 11 Hallmark residue: L, S, V, M, W, F, T, Q, E, A, R, G, K, Y,0.35 4 N, P, I; preferably L 12 V, I V, A, L, M, E, G, T 0.11 2 13 Q, K,R Q, L, R, H, P, E, K, T, S, V, D, G, A, N, M 0.46 3 14 P A, P, T, V, S,D, F, N, I, E, L, R, G, Y, Q, H 0.92 5 15 G G, E 0 1 16 G, R G, D, E, A,S, N, V, R, K, T, P, C, L 0.47 4 17 S S, F, P, Y, T, A, C, R, N 0.14 218 L L, V, R, M, P, Q, S, A, T, K, H 0.06 1 19 R, K R, T, K, S, N, G, A,I, L, Q, F, E, V, M 0.36 4 20 L L, F, V, I, P, H, S 0.18 3 21 S S, A, T,P, F, V, H, D, R, L, I, G 0.13 3 22 C C, W 0 1 23 A, T A, V, T, E, S, L,G, I, K, Q, R, D, F, N, P, M 0.88 5 24 A A, D, V, T, H, Y, P, G, S, F,L, I, N, Q, E, R 0.78 9 25 S S, P, T, A, F, L, N, Y, R, H, D, V, I, W,G, K, Q, C 0.2 2 26 G G, E, R, V, T, A, S, K, D, L, I, Q, N, F, Y, M,0.45 6 W, P, H 27 F R, F, S, P, L, G, I, N, T, D, H, V, E, A, Y, K, M,1.89 12 Q, W, C 28 T T, I, S, A, P, F, D, N, V, R, M, L, G, Y, K, E, H,1.29 12 W, Q 29 F, V F, L, S, V, I, A, W, Y, G, D, R, T, P, N, E, M,1.23 11 H, Q, K, C 30 S, D, G S, D, N, G, R, T, A, E, I, Y, K, V, H, L,F, W, 1.55 12 M, P, C, Q

TABLE B-5 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table B-2) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0 1 37Hallmark residue: F⁽¹⁾, Y, V, L, A, H, S, I, W, C, N, G, D, T, P, 1.1 7preferably F⁽¹⁾ or Y 38 R R, H, C, P, Y, L, V 0.01 1 39 Q Q, E, R, H, L,A, S, K, P, V, T, D 0.22 3 40 A A, V, T, P, G, S, D, I, L, R, N, F, Y,C, E, H 0.55 6 41 P, S, T P, S, A, L, T, Q, R, V, D, G, I, H 0.18 3 42 GG, E, A, R, D, V, W, T, Q, K, L, N, H, M 0.1 2 43 K K, N, Q, E, R, T, L,S, M, D, G, A, V, H, I, F, P 0.45 7 44 Hallmark residue: E⁽³⁾, Q⁽³⁾,G⁽²⁾, D, A, K, R, L, P, S, V, H, T, 1.11 4 N, W, M, I; preferably G⁽²⁾,E⁽³⁾ or Q⁽³⁾; most preferably G⁽²⁾ or Q⁽³⁾. 45 Hallmark residue: L⁽²⁾,R⁽³⁾, P, H, F, G, Q, S, E, T, Y, C, I, D, 0.56 3 V; preferably L⁽²⁾ orR⁽³⁾ 46 E, V E, D, A, Q, V, M, K, T, G, R, S, N, I, L, F 0.42 4 47Hallmark residue: F⁽¹⁾, L⁽¹⁾ or W⁽²⁾ G, I, S, A, V, M, R, Y, E, 1.64 11P, T, C, H, K, Q, N, D; preferably W⁽²⁾, L⁽¹⁾ or F⁽¹⁾ 48 V V, I, L, A,T, Q, F, M, G, E, R 0.35 5 49 S, A, G A, S, G, T, V, L, C, I, F, P, E,Y, M, D, R 0.89 5

TABLE B-6 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table B-2) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 66 R R 0 1 67 F F,S, L, V, I, C, A, Y, M, G 0.1 1 68 T T, A, S, I, F, V, P, N, G, R, K, M,D, L, W, Q 0.34 4 69 I I, V, M, T, L, A, F, P, S, G, N 0.5 5 70 S S, T,A, F, P, V, Y, L, D, G, N, H, W, E, C 0.22 4 71 R R, S, K, G, T, I, W,A, N, V, E, L, M, F, D, 0.61 7 Q, C 72 D, E D, N, E, G, V, A, H, L, S,T, I, Q, F, P, Y, R 0.34 4 73 N, D, G N, D, S, K, I, Y, G, T, H, R, A,V, F, L, E, 0.65 9 M, P, C 74 A, S A, T, V, S, F, G, D, P, N, I, R, L,Y, H, E, 0.8 8 Q, K, W, M 75 K K, N, E, R, Q, A, G, T, M, S, L, D, V, W,Y, I 0.71 6 76 N, S N, K, S, R, D, T, H, G, E, A, Y, I, M, Q, L, 0.66 7W, P, F, V 77 S, T, I T, A, M, S, R, I, V, L, P, E, N, K, G, W, Q 0.72 778 L, A V, L, A, M, I, G, T, F, W, Q, S, E, N, H 1.11 6 79 Y, H Y, F, D,S, H, N, T, A, L, W, V, C, G, E, I, 0.68 8 P, R 80 L L, M, V, P, F 0.052 81 Q Q, E, R, H, L, D, T, G, K, P, A, I, S, N, Y, 0.38 4 V, M 82 M M,I, L, V, A, T, S, K 0.12 3 82a N, G N, S, D, T, E, H, K, I, A, G, R, Y,L, V, F, Q 0.77 5 82b S S, N, T, G, H, D, R, A, K, I, M, V, F, E, P, Y,0.72 8 C, L 82c L L, V, M, P, A, T, G 0.08 2 83 Hallmark residue: R,K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I, V, G, M, L, A, D, 0.66 6 Y, H; preferably Kor R; most preferably K 84 Hallmark residue: P⁽⁵⁾, S, H, L, A, V, I, T,F, D, R, Y, N, Q, G, 0.85 7 E; preferably P 85 E, G E, D, G, A, Q, V, S,N, K, T, R, L 0.27 3 86 D D, E, G, N 0.02 1 87 T, M T, S, A, M, R, P, K,E 0.15 3 88 A A, G, S, D, N, T, P, V 0.23 2 89 V, L V, I, L, E, A, R, T,D, F, M, N, S, K, G, Q, H 0.71 7 90 Y Y, H, F, N 0 1 91 Y, H Y, F, R, S,H, T, I, V, L, N, D, C, Q, W, A, E, M 0.6 7 92 C C, R, P 0 1 93 A, K, TA, N, T, K, G, V, R, Y, S, H, W, L, F, Q, M, I, 1.33 10 E, C, D 94 K, R,T A, K, V, T, R, L, G, S, D, Q, I, M, F, Y, N, E, 1.55 12 H, P, C, W

TABLE B-7 Non-limiting examples of amino acid residues in FR4 (for thefootnotes, see the footnotes to Table B-2) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 103 Hallmarkresidue: W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V, 0.54 6 Q, P⁽⁶⁾, E,C; preferably W 104 Hallmark residue: G, A, S, T, D, P, N, E, C, L;preferably G 0.13 3 105 Q, R Q, K, H, R, P, E, L, T, N, S, V, A, M, G0.52 5 106 G G, R, E 0 1 107 T T, Q, I, A, S, N, R, V, D 0.24 3 108Hallmark residue: Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H; 0.3 4 preferably Qor L⁽⁷⁾ 109 V V, I, L 0 1 110 T T, S, N, A, I, F 0.01 1 111 V V, I, A0.01 1 112 S S, T, P, F, A 0.01 1 113 S S, T, A, L, P, F, E, V 0.04 1

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can be defined as an immunoglobulin sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4    -   in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) one or more of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table B-2;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In particular, a Nanobody of the invention can be an immunoglobulinsequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4    -   in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:        i) (preferably) one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2 (it being understood that V_(HH)        sequences will contain one or more Hallmark residues; and that        partially humanized Nanobodies will usually, and preferably,        [still] contain one or more Hallmark residues [although it is        also within the scope of the invention to provide—where suitable        in accordance with the invention—partially humanized Nanobodies        in which all Hallmark residues, but not one or more of the other        amino acid residues, have been humanized]; and that in fully        humanized Nanobodies, where suitable in accordance with the        invention, all amino acid residues at the positions of the        Hallmark residues will be amino acid residues that occur in a        human V_(H)3 sequence. As will be clear to the skilled person        based on the disclosure herein that such V_(HH) sequences, such        partially humanized Nanobodies with at least one Hallmark        residue, such partially humanized Nanobodies without Hallmark        residues and such fully humanized Nanobodies all form aspects of        this invention);        and in which:

-   ii) said immunoglobulin sequence has at least 80% amino acid    identity with at least one of the immunoglobulin sequences of SEQ ID    NO's: 1 to 22, in which for the purposes of determining the degree    of amino acid identity, the amino acid residues that form the CDR    sequences (indicated with X in the sequences of SEQ ID NO's: 1    to 22) are disregarded;    and in which:

-   iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

TABLE B-8 Representative immunoglobulin sequences for Nanobodies of theKERE, GLEW and P, R, S 103 group. The CDR's are indicated with XXXX KEREsequence no. 1 SEQ ID NO: 1 EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFX XXXXWGQGTQVTVSS KEREsequence no. 2 SEQ ID NO: 2 QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTASNRGYLHMNNLTPEDTAVYYCAA XXXXXWGQGTQVTVSS KEREsequence no. 3 SEQ ID NO: 3 AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTISRDNAKNMVYLRMNSLIPEDAAVYSCAA XXXXXWGQGTLVTVSS KEREsequence no. 4 SEQ ID NO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTISRDDAKNTVWLHGSTLKPEDTAVYYCAA XXXXXWGQGTQVTVSS KEREsequence no. 5 SEQ ID NO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKI XXXXXWGQGTQVTVSS KEREsequence no. 6 SEQ ID NO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTISSEKDKNSVYLQMNSLKPEDTALYICAG XXXXXWGRGTQVTVSS KEREsequence no. 7 SEQ ID NO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTIARDSTKDTFCLQMNNLKPEDTAVYYCYA XXXXXWGQGTQVTVSS KEREsequence no. 8 SEQ ID NO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTISTDNAKNTVHLLMNRVNAEDTALYYCAV XXXXXWGRGTRVTVSS KEREsequence no. 9 SEQ ID NO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTISGDNAKRAIYLQMNNLKPDDTAVYYCNR XXXXXWGQGTQVTVSP KEREsequence no. SEQ ID NO: 10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGK10 EREFVAXXXXXRFTISRNATKNTLTLRMDSLKPEDTAVYYCAAX XXXXWGQGTQVTVSS KEREsequence no. SEQ ID NO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPG 11EKREFVAXXXXXRFTIARENAGNMVYLQMNNLKPDDTALYTCAA XXXXXWGRGTQVTVSS KEREsequence no. SEQ ID NO: 12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPG 12KERVFLAXXXXXRFTISRDSAKNMMYLQMNNLKPQDTAVYYCA AXXXXXWGQGTQVTVSS KEREsequence no. SEQ ID NO: 13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPW 13QERDFVAXXXXXRFTISRDNYKDTVLLEMNFLKPEDTAIYYCAAX XXXXWGQGTQVTVSS KEREsequence no. SEQ ID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGR14 DREFVAXXXXXRFTVSRDSAENTVALQMNSLKPEDTAVYYCAA XXXXXWGQGTRVTVSS KEREsequence no. SEQ ID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPG 15KEREAVSXXXXXRFTISRDYAGNTAFLQMDSLKPEDTGVYYCAT XXXXXWGQGTQVTVSS KEREsequence no. SEQ ID NO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPG 16KEREFVAXXXXXRFTVSRDNGKNTAYLRMNSLKPEDTADYYCA VXXXXXLGSGTQVTVSS GLEWsequence no. 1 SEQ ID NO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTISRDNAKNTLYLQMNSLKPEDTAVYYCVK XXXXXGSQGTQVTVSS GLEWsequence no. 2 SEQ ID NO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQ RXXXXXRGQGTQVTVSS GLEWsequence no. 3 SEQ ID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTISRDNAKNTLYLEMNSLSPEDTAMYYCGR XXXXXRSKGIQVTVSS P, R, S 103sequence SEQ ID NO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPG no. 1KEREFVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA XXXXXRGQGTQVTVSS P, R, S103 sequence SEQ ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGno. 2 KGLEWVGXXXXXRFTISRDNAKNMLYLHLNNLKSEDTAVYYCR RXXXXXLGQGTQVTVSS P,R, S 103 sequence SEQ ID NO: 22EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPG no. 3KAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQ RXXXXXRGQGTQVTVSS

In particular, a Nanobody of the invention of the KERE group can be animmunoglobulin sequence with the (general) structure

-   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4    in which:-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-9 Representative FW1 sequences for Nanobodies of the KERE-group.KERE FW1 SEQ ID QVQRVESGGGLVQAGGSLRLSCAASG sequence no. 1 NO: 23 RTSSKERE FW1 SEQ ID QVQLVESGGGLVQTGDSLSLSCSASG sequence no. 2 NO: 24 RTFSKERE FW1 SEQ ID QVKLEESGGGLVQAGDSLRLSCAATG sequence no. 3 NO: 25 RAFGKERE FW1 SEQ ID AVQLVESGGGLVQPGESLGLSCVASG sequence no. 4 NO: 26 RDFVKERE FW1 SEQ ID EVQLVESGGGLVQAGGSLRLSCEVLG sequence no. 5 NO: 27 RTAGKERE FW1 SEQ ID QVQLVESGGGWVQPGGSLRLSCAASE sequence no. 6 NO: 28 TILSKERE FW1 SEQ ID QVQLVESGGGTVQPGGSLNLSCVASG sequence no. 7 NO: 29 NTFNKERE FW1 SEQ ID EVQLVESGGGLAQPGGSLQLSCSAPG sequence no. 8 NO: 30 FTLDKERE FW1 SEQ ID AQELEESGGGLVQAGGSLRLSCAASG sequence no. 9 NO: 31 RTFNand in which:

-   iii) FR2 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-10 Representative FW2 sequences for Nanobodies of theKERE-group. KERE FW2 sequence no. 1 SEQ ID WFRQAPGKEREFVA NO: 41 KEREFW2 sequence no. 2 SEQ ID WFRQTPGREREFVA NO: 42 KERE FW2 sequence no. 3SEQ ID WYRQAPGKQREMVA NO: 43 KERE FW2 sequence no. 4 SEQ IDWYRQGPGKQRELVA NO: 44 KERE FW2 sequence no. 5 SEQ ID WIRQAPGKEREGVS NO:45 KERE FW2 sequence no. 6 SEQ ID WFREAPGKEREGIS NO: 46 KERE FW2sequence no. 7 SEQ ID WYRQAPGKERDLVA NO: 47 KERE FW2 sequence no. 8 SEQID WFRQAPGKQREEVS NO: 48 KERE FW2 sequence no. 9 SEQ ID WFRQPPGKVREFVGNO: 49and in which:

-   iv) FR3 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-11 Representative FW3 sequences for Nanobodies of theKERE-group. KERE FW3 SEQ ID RFTISRDNAKNTVYLQMNSLKPEDTAV sequence no. 1NO: 50 YRCYF KERE FW3 SEQ ID RFAISRDNNKNTGYLQMNSLEPEDTA sequence no. 2NO: 51 VYYCAA KERE FW3 SEQ ID RFTVARNNAKNTVNLEMNSLKPEDTA sequence no. 3NO: 52 VYYCAA KERE FW3 SEQ ID RFTISRDIAKNTVDLLMNNLEPEDTAV sequence no. 4NO: 53 YYCAA KERE FW3 SEQ ID RLTISRDNAVDTMYLQMNSLKPEDTA sequence no. 5NO: 54 VYYCAA KERE FW3 SEQ ID RFTISRDNAKNTVYLQMDNVKPEDTAI sequence no. 6NO: 55 YYCAA KERE FW3 SEQ ID RFTISKDSGKNTVYLQMTSLKPEDTAV sequence no. 7NO: 56 YYCAT KERE FW3 SEQ ID RFTISRDSAKNMMYLQMNNLKPQDTA sequence no. 8NO: 57 VYYCAA KERE FW3 SEQ ID RFTISRENDKSTVYLQLNSLKPEDTAV sequence no. 9NO: 58 YYCAA KERE FW3 SEQ ID RFTISRDYAGNTAYLQMNSLKPEDTG sequence no. 10NO: 59 VYYCATand in which:

-   v) FR4 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-12 Representative FW4 sequences for Nanobodies of theKERE-group. KERE FW4 sequence no. 1 SEQ ID WGQGTQVTVSS NO: 60 KERE FW4sequence no. 2 SEQ ID WGKGTLVTVSS NO: 61 KERE FW4 sequence no. 3 SEQ IDRGQGTRVTVSS NO: 62 KERE FW4 sequence no. 4 SEQ ID WGLGTQVTISS NO: 63and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

With regard to framework 1, it will be clear to the skilled person that,when an immunoglobulin sequence as outlined above is generated byexpression of a nucleotide sequence, the first four immunoglobulinsequences (i.e. amino acid residues 1-4 according to the Kabatnumbering) may often be determined by the primer(s) that have been usedto generate said nucleic acid. Thus, for determining the degree of aminoacid identity, the first four amino acid residues are preferablydisregarded.

Also, with regard to framework 1, and although amino acid positions 27to 30 are according to the Kabat numbering considered to be part of theframework regions (and not the CDR's), it has been found by analysis ofa database of more than 1000 V_(HH) sequences that the positions 27 to30 have a variability (expressed in terms of V_(HH) entropy and V_(HH)variability—see Tables B-4 to B-7) that is much greater than thevariability on positions 1 to 26. Because of this, for determining thedegree of amino acid identity, the amino acid residues at positions 27to 30 are preferably also disregarded.

In view of this, a Nanobody of the KERE class may be an immunoglobulinsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an immunoglobulin sequence that, on positions 5 to 26 of    the Kabat numbering, has at least 80% amino acid identity with at    least one of the following immunoglobulin sequences:

TABLE B-13 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. KERE FW1 SEQ ID VESGGGLVQPGGSLRLSCAASGsequence no. 10 NO: 32 KERE FW1 SEQ ID VDSGGGLVQAGDSLKLSCALTG sequenceno. 11 NO: 33 KERE FW1 SEQ ID VDSGGGLVQAGDSLRLSCAASG sequence no. 12 NO:34 KERE FW1 SEQ ID VDSGGGLVEAGGSLRLSCQVSE sequence no. 13 NO: 35 KEREFW1 SEQ ID QDSGGGSVQAGGSLKLSCAASG sequence no. 14 NO: 36 KERE FW1 SEQ IDVQSGGRLVQAGDSLRLSCAASE sequence no. 15 NO: 37 KERE FW1 SEQ IDVESGGTLVQSGDSLKLSCASST sequence no. 16 NO: 38 KERE FW1 SEQ IDMESGGDSVQSGGSLTLSCVASG sequence no. 17 NO: 39 KERE FW1 SEQ IDQASGGGLVQAGGSLRLSCSASV sequence no. 18 NO: 40and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the KERE-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

A Nanobody of the GLEW class may be an immunoglobulin sequence that iscomprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;-   ii) FR1 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-14 Representative FW1 sequences for Nanobodies of theGLEW-group. GLEW FW1 SEQ ID QVQLVESGGGLVQPGGSLRLSCAAS sequence no. 1 NO:64 GFTFS GLEW FW1 SEQ ID EVHLVESGGGLVRPGGSLRLSCAAFG sequence no. 2 NO:65 FIFK GLEW FW1 SEQ ID QVKLEESGGGLAQPGGSLRLSCVASG sequence no. 3 NO: 66FTFS GLEW FW1 SEQ ID EVQLVESGGGLVQPGGSLRLSCVCVS sequence no. 4 NO: 67SGCT GLEW FW1 SEQ ID EVQLVESGGGLALPGGSLTLSCVFSG sequence no. 5 NO: 68STFSand in which:

-   iii) FR2 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-15 Representative FW2 sequences for Nanobodies of theGLEW-group. GLEW FW2 sequence no. 1 SEQ ID WVRQAPGKVLEWVS NO: 72 GLEWFW2 sequence no. 2 SEQ ID WVRRPPGKGLEWVS NO: 73 GLEW FW2 sequence no. 3SEQ ID WVRQAPGMGLEWVS NO: 74 GLEW FW2 sequence no. 4 SEQ IDWVRQAPGKEPEWVS NO: 75 GLEW FW2 sequence no. 5 SEQ ID WVRQAPGKDQEWVS NO:76 GLEW FW2 sequence no. 6 SEQ ID WVRQAPGKAEEWVS NO: 77 GLEW FW2sequence no. 7 SEQ ID WVRQAPGKGLEWVA NO: 78 GLEW FW2 sequence no. 8 SEQID WVRQAPGRATEWVS NO: 79and in which:

-   iv) FR3 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-16 Representative FW3 sequences for Nanobodies of the GLEW-group. GLEW FW3 SEQ ID RFTISRDNAKNTLYLQMNSLKPEDTAV sequence no. 1 NO: 80YYCVK GLEW FW3 SEQ ID RFTISRDNARNTLYLQMDSLIPEDTAL sequence no. 2 NO: 81YYCAR GLEW FW3 SEQ ID RFTSSRDNAKSTLYLQMNDLKPEDTA sequence no. 3 NO: 82LYYCAR GLEW FW3 SEQ ID RFIISRDNAKNTLYLQMNSLGPEDTAM sequence no. 4 NO: 83YYCQR GLEW FW3 SEQ ID RFTASRDNAKNTLYLQMNSLKSEDTA sequence no. 5 NO: 84RYYCAR GLEW FW3 SEQ ID RFTISRDNAKNTLYLQMDDLQSEDTA sequence no. 6 NO: 85MYYCGRand in which:

-   v) FR4 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-17 Representative FW4 sequences for Nanobodies of theGLEW-group. GLEW FW4 sequence no. 1 SEQ ID GSQGTQVTVSS NO: 86 GLEW FW4sequence no. 2 SEQ ID LRGGTQVTVSS NO: 87 GLEW FW4 sequence no. 3 SEQ IDRGQGTLVTVSS NO: 88 GLEW FW4 sequence no. 4 SEQ ID RSRGIQVTVSS NO: 89GLEW FW4 sequence no. 5 SEQ ID WGKGTQVTVSS NO: 90 GLEW FW4 sequence no.6 SEQ ID WGQGTQVTVSS NO: 91and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an immunoglobulinsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;    and in which:-   ii) FR1 is an immunoglobulin sequence that, on positions 5 to 26 of    the Kabat numbering, has at least 80% amino acid identity with at    least one of the following immunoglobulin sequences:

TABLE B-18 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. GLEW FW1 sequence SEQ IDVESGGGLVQPGGSLRLSCAASG no. 6 NO: 69 GLEW FW1 sequence SEQ IDEESGGGLAQPGGSLRLSCVASG no. 7 NO: 70 GLEW FW1 sequence SEQ IDVESGGGLALPGGSLTLSCVFSG no. 8 NO: 71and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the GLEW-class;    and in which:-   iv) CDR1. CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein. Inthe above Nanobodies, one or more of the further Hallmark residues arepreferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an immunoglobulin sequencethat is comprised of four framework regions/sequences interrupted bythree complementarity determining regions/sequences, in which

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or 5, and more preferably R;    and in which:-   iii) FR1 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-19 Representative FW1 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW1 SEQ ID AVQLVESGGGLVQAGGSLRLSCAAS sequence no.1 NO: 92 GRTFS P, R, S 103 FW1 SEQ ID QVQLQESGGGMVQPGGSLRLSCAA sequenceno. 2 NO: 93 SGFDFG P, R, S 103 FW1 SEQ ID EVHLVESGGGLVRPGGSLRLSCAAFsequence no. 3 NO: 94 GFIFK P, R, S 103 FW1 SEQ IDQVQLAESGGGLVQPGGSLKLSCAAS sequence no. 4 NO: 95 RTIVS P, R, S 103 FW1SEQ ID QEHLVESGGGLVDIGGSLRLSCAASE sequence no. 5 NO: 96 RIFS P, R, S 103FW1 SEQ ID QVKLEESGGGLAQPGGSLRLSCVAS sequence no. 6 NO: 97 GFTFS P, R, S103 FW1 SEQ ID EVQLVESGGGLVQPGGSLRLSCVCV sequence no. 7 NO: 98 SSGCT P,R, S 103 FW1 SEQ ID EVQLVESGGGLALPGGSLTLSCVFS sequence no. 8 NO: 99GSTFSand in which

-   iv) FR2 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-20 Representative FW2 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW2 sequence SEQ ID WFRQAPGKEREFVA no. 1 NO: 102P, R, S 103 FW2 sequence SEQ ID WVRQAPGKVLEWVS no. 2 NO: 103 P, R, S 103FW2 sequence SEQ ID WVRRPPGKGLEWVS no. 3 NO: 104 P, R, S 103 FW2sequence SEQ ID WIRQAPGKEREGVS no. 4 NO: 105 P, R, S 103 FW2 sequenceSEQ ID WVRQYPGKEPEWVS no. 5 NO: 106 P, R, S 103 FW2 sequence SEQ IDWFRQPPGKEHEFVA no. 6 NO: 107 P, R, S 103 FW2 sequence SEQ IDWYRQAPGKRTELVA no. 7 NO: 108 P, R, S 103 FW2 sequence SEQ IDWLRQAPGQGLEWVS no. 8 NO: 109 P, R, S 103 FW2 sequence SEQ IDWLRQTPGKGLEWVG no. 9 NO: 110 P, R, S 103 FW2 sequence SEQ IDWVRQAPGKAEEFVS no. 10 NO: 111and in which:

-   v) FR3 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-21 Representative FW3 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW3 SEQ ID RFTISRDNAKNTVYLQMNSLKPEDTA sequenceno. 1 NO: 112 VYYCAA P, R, S 103 FW3 SEQ ID RFTISRDNARNTLYLQMDSLIPEDTAsequence no. 2 NO: 113 LYYCAR P, R, S 103 FW3 SEQ IDRFTISRDNAKNEMYLQMNNLKTEDTG sequence no. 3 NO: 114 VYWCGA P, R, S 103 FW3SEQ ID RFTISSDSNRNMIYLQMNNLKPEDTA sequence no. 4 NO: 115 VYYCAA P, R, S103 FW3 SEQ ID RFTISRDNAKNMLYLHLNNLKSEDTA sequence no. 5 NO: 116 VYYCRRP, R, S 103 FW3 SEQ ID RFTISRDNAKKTVYLRLNSLNPEDTA sequence no. 6 NO: 117VYSCNL P, R, S 103 FW3 SEQ ID RFKISRDNAKKTLYLQMNSLGPEDTA sequence no. 7NO: 118 MYYCQR P, R, S 103 FW3 SEQ ID RFTVSRDNGKNTAYLRMNSLKPEDTAsequence no. 8 NO: 119 DYYCAVand in which:

-   vi) FR4 is an immunoglobulin sequence that has at least 80% amino    acid identity with at least one of the following immunoglobulin    sequences:

TABLE B-22 Representative FW4 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW4 sequence SEQ ID RGQGTQVTVSS no. 1 NO: 120 P,R, S 103 FW4 sequence SEQ ID LRGGTQVTVSS no. 2 NO: 121 P, R, S 103 FW4sequence SEQ ID GNKGTLVTVSS no. 3 NO: 122 P, R, S 103 FW4 sequence SEQID SSPGTQVTVSS no. 4 NO: 123 P, R, S 103 FW4 sequence SEQ ID SSQGTLVTVSSno. 5 NO: 124 P, R, S 103 FW4 sequence SEQ ID RSRGIQVTVSS no. 6 NO: 125and in which:

-   vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the P, R, S 103 class may be animmunoglobulin sequence that is comprised of four frameworkregions/sequences interrupted by three complementarity determiningregions/sequences, in which:

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an immunoglobulin sequence that, on positions 5 to 26 of    the Kabat numbering, has at least 80% amino acid identity with at    least one of the following immunoglobulin sequences:

TABLE B-23 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the P, R, S 103-group. P, R, S 103 FW1 SEQ IDVESGGGLVQAGGSLRLSCAASG sequence no. 9 NO: 100 P, R, S 103 FW1 SEQ IDAESGGGLVQPGGSLKLSCAASR sequence no. 10 NO: 101and in which:

-   iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of    Nanobodies of the P, R, S 103 class;    and in which:-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody as described above, in which the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the immunoglobulin sequences of SEQ IDNO's: 705 to 788, more preferably SEQ ID NO's 726 to 750, 753 to 758,762 to 764, 772 to 773, 775, or 778 to 780, more preferred SEQ ID NO's732, 773 or 778 (see Table A-1). This degree of amino acid identity canfor example be determined by determining the degree of amino acididentity (in a manner described herein) between said Nanobody and one ormore of the sequences of SEQ ID NO's: 705 to 788, more preferably SEQ IDNO's 726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or 778 to 780,more preferred SEQ ID NO's 732, 773 or 778 (see Table A-1), in which theamino acid residues that form the framework regions are disregarded.Such Nanobodies can be as further described herein.

As already mentioned herein, another preferred but non-limiting aspectof the invention relates to a Nanobody with an immunoglobulin sequencethat is chosen from the group consisting of SEQ ID NO's: 705 to 788,more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or 778 (seeTable A-1) or from the group consisting of from immunoglobulin sequencesthat have more than 80%, preferably more than 90%, more preferably morethan 95%, such as 99% or more sequence identity (as defined herein) withat least one of the immunoglobulin sequences of SEQ ID NO's: 705 to 788,more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or 778 (seeTable A-1).

-   -   Also, in the above Nanobodies:

-   i) any amino acid substitution (when it is not a humanizing    substitution as defined herein) is preferably, and compared to the    corresponding immunoglobulin sequence of SEQ ID NO's: 705 to 788,    more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772    to 773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or    778 (see Table A-1), a conservative amino acid substitution, (as    defined herein);    and/or:

-   ii) its immunoglobulin sequence preferably contains either only    amino acid substitutions, or otherwise preferably no more than 5,    preferably no more than 3, and more preferably only 1 or 2 amino    acid deletions or insertions, compared to the corresponding    immunoglobulin sequence of SEQ ID NO's: 705 to 788, more preferably    SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or    778 to 780, more preferred SEQ ID NO's 732, 773 or 778 (see Table    A-1);    and/or

-   iii) the CDR's may be CDR's that are derived by means of affinity    maturation, for example starting from the CDR's of to the    corresponding immunoglobulin sequence of SEQ ID NO's: 705 to 788,    more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772    to 773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or    778 (see Table A-1).

Preferably, the CDR sequences and FR sequences in the Nanobodies of theinvention are such that the Nanobodies of the invention (andpolypeptides of the invention comprising the same):

-   -   bind to ion channels such as e.g. P2X7 with a dissociation        constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and        preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably        10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association constant        (K_(A)) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷        to 10¹² liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that they:    -   bind to ion channels such as e.g. P2X7 with a k_(on)-rate of        between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³        M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and        10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to ion channels such as e.g. P2X7 with a k_(off) rate        between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near        irreversible complex with a t_(1/2) of multiple days),        preferably between 10² s′¹ and 10⁶ s¹ more preferably between        10′³ s′¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, CDR sequences and FR sequences present in the Nanobodies ofthe invention are such that the Nanobodies of the invention will bind toion channels such as e.g. P2X7 with an affinity less than 500 nM,preferably less than 200 nM, more preferably less than 10 nM, such asless than 500 pM.

According to one non-limiting aspect of the invention, a Nanobody may beas defined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human V_(H) domain, and in particular compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a Nanobody may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. Usually, a Nanobody will have at least one such amino aciddifference with a naturally occurring V_(H) domain in at least one ofFR2 and/or FR4, and in particular at at least one of the Hallmarkresidues in FR2 and/or FR4 (again, including those at positions 108, 103and/or 45).

Also, a humanized Nanobody of the invention may be as defined herein,but with the proviso that it has at least “one amino acid difference”(as defined herein) in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring V_(HH)domain. More specifically, according to one non-limiting aspect of theinvention, a humanized Nanobody may be as defined herein, but with theproviso that it has at least “one amino acid difference” (as definedherein) at at least one of the Hallmark residues (including those atpositions 108, 103 and/or 45) compared to the corresponding frameworkregion of a naturally occurring V_(HH) domain. Usually, a humanizedNanobody will have at least one such amino acid difference with anaturally occurring V_(HH) domain in at least one of FR2 and/or FR4, andin particular at at least one of the Hallmark residues in FR2 and/or FR4(again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scopeof the invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the Nanobodies of the invention as defined herein, and inparticular analogs of the Nanobodies of SEQ ID NO's 705 to 788, morepreferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773,775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or 778 (seeTable A-1). Thus, according to one aspect of the invention, the term“Nanobody of the invention” in its broadest sense also covers suchanalogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the Nanobodies of theinvention as defined herein. Such substitutions, insertions or deletionsmay be made in one or more of the framework regions and/or in one ormore of the CDR's. When such substitutions, insertions or deletions aremade in one or more of the framework regions, they may be made at one ormore of the Hallmark residues and/or at one or more of the otherpositions in the framework residues, although substitutions, insertionsor deletions at the Hallmark residues are generally less preferred(unless these are suitable humanizing substitutions as describedherein).

By means of non-limiting examples, a substitution may for example be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see Tables B-4 toB-7 for some non-limiting examples of such substitutions), although theinvention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the Nanobody of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the Nanobody of theinvention (i.e. to the extent that the Nanobody is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express theNanobody or polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables B-4 to B-7 above, some amino acid residuesin the framework regions are more conserved than others. Generally,although the invention in its broadest sense is not limited thereto, anysubstitutions, deletions or insertions are preferably made at positionsthat are less conserved. Also, generally, amino acid substitutions arepreferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to ion channels suchas e.g. P2X7 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein for the Nanobodiesof the invention.

The analogs are preferably also such that they retain the favourableproperties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree ofsequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, such as at least 95% or 99% or more; and/orpreferably have at most 20, preferably at most 10, even more preferablyat most 5, such as 4, 3, 2 or only 1 amino acid difference (as definedherein), with one of the Nanobodies of SEQ ID NOs: 705 to 788, morepreferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773,775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or 778 (seeTable A-1).

Also, the framework sequences and CDR's of the analogs are preferablysuch that they are in accordance with the preferred aspects definedherein. More generally, as described herein, the analogs will have (a) aQ at position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably an E at position 44, and more preferably Eat position 44 and R at position 45; and/or (c) P, R or S at position103.

One preferred class of analogs of the Nanobodies of the inventioncomprise Nanobodies that have been humanized (i.e. compared to thesequence of a naturally occurring Nanobody of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, for example from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparison between the sequence of a Nanobody and the sequence of anaturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resultinghumanized Nanobodies still retain the favourable properties ofNanobodies as defined herein, and more preferably such that they are asdescribed for analogs in the preceding paragraphs. A skilled person willgenerally be able to determine and select suitable humanizingsubstitutions or suitable combinations of humanizing substitutions,based on the disclosure herein and optionally after a limited degree ofroutine experimentation, which may for example involve introducing alimited number of possible humanizing substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the inventionmay become more “humanlike”, while still retaining the favorableproperties of the Nanobodies of the invention as described herein. As aresult, such humanized Nanobodies may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favourable propertiesprovided by the humanizing substitutions on the one hand and thefavourable properties of naturally occurring V_(HH) domains on the otherhand.

The Nanobodies of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined herein) or at one or more other framework residues (i.e.non-Hallmark residues) or any suitable combination thereof. Onepreferred humanizing substitution for Nanobodies of the “P, R, S-103group” or the “KERE group” is Q108 into L108. Nanobodies of the “GLEWclass” may also be humanized by a Q108 into L108 substitution, providedat least one of the other Hallmark residues contains a camelid(camelizing) substitution (as defined herein). For example, as mentionedabove, one particularly preferred class of humanized Nanobodies has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se, for example using oneor more of the techniques mentioned on pages 103 and 104 of WO08/020079.

As mentioned there, it will be also be clear to the skilled person thatthe Nanobodies of the invention (including their analogs) can bedesigned and/or prepared starting from human V_(H) sequences (i.e.immunoglobulin sequences or the corresponding nucleotide sequences),such as for example from human V_(H)3 sequences such as DP-47, DP-51 orDP-29, i.e. by introducing one or more camelizing substitutions (i.e.changing one or more amino acid residues in the immunoglobulin sequenceof said human V_(H) domain into the amino acid residues that occur atthe corresponding position in a V_(HH) domain), so as to provide thesequence of a Nanobody of the invention and/or so as to confer thefavourable properties of a Nanobody to the sequence thus obtained.Again, this can generally be performed using the various methods andtechniques referred to in the previous paragraph, using animmunoglobulin sequence and/or nucleotide sequence for a human V_(H)domain as a starting point.

Some preferred, but non-limiting camelizing substitutions can be derivedfrom Tables B-4-B-7. It will also be clear that camelizing substitutionsat one or more of the Hallmark residues will generally have a greaterinfluence on the desired properties than substitutions at one or more ofthe other amino acid positions, although both and any suitablecombination thereof are included within the scope of the invention. Forexample, it is possible to introduce one or more camelizingsubstitutions that already confer at least some the desired properties,and then to introduce further camelizing substitutions that eitherfurther improve said properties and/or confer additional favourableproperties. Again, the skilled person will generally be able todetermine and select suitable camelizing substitutions or suitablecombinations of camelizing substitutions, based on the disclosure hereinand optionally after a limited degree of routine experimentation, whichmay for example involve introducing a limited number of possiblecamelizing substitutions and determining whether the favourableproperties of Nanobodies are obtained or improved (i.e. compared to theoriginal V₁₁ domain).

Generally, however, such camelizing substitutions are preferably suchthat the resulting an immunoglobulin sequence at least contains (a) a Qat position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably also an E at position 44, and morepreferably E at position 44 and R at position 45; and/or (c) P, R or Sat position 103; and optionally one or more further camelizingsubstitutions. More preferably, the camelizing substitutions are suchthat they result in a Nanobody of the invention and/or in an analogthereof (as defined herein), such as in a humanized analog and/orpreferably in an analog that is as defined in the preceding paragraphs.

Nanobodies can also be derived from V_(H) domains by the incorporationof substitutions that are rare in nature, but nonetheless, structurallycompatible with the VH domain fold. For example, but without beinglimiting, these substitutions may include on or more of the following:Gly at position 35, Ser, Val or Thr at position 37, Ser, Thr, Arg, Lys,His, Asp or Glu at position 39, Glu or His at position 45, Trp, Leu,Val, Ala, Thr, or Glu at position 47, S or R at position 50. (Barthelemyet al. J Biol Chem. 2008 Feb. 8; 283(6):3639-54. Epub 2007 Nov. 28)

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the Nanobodies of the invention asdefined herein, and in particular parts or fragments of the Nanobodiesof SEQ ID NO's: 705 to 788, more preferably SEQ ID NO's 726 to 750, 753to 758, 762 to 764, 772 to 773, 775, or 778 to 780, more preferred SEQID NO's 732, 773 or 778 (see Table A-1). Thus, according to one aspectof the invention, the term “Nanobody of the invention” in its broadestsense also covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention(including analogs thereof) have immunoglobulin sequences in which,compared to the immunoglobulin sequence of the corresponding full lengthNanobody of the invention (or analog thereof), one or more of the aminoacid residues at the N-terminal end, one or more amino acid residues atthe C-terminal end, one or more contiguous internal amino acid residues,or any combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to ionchannels such as e.g. P2X7 with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined hereinfor the Nanobodies of the invention.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theimmunoglobulin sequence of the corresponding full length Nanobody of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect,such a part or fragment comprises at least CDR3, such as FR3, CDR3 andFR4 of the corresponding full length Nanobody of the invention, i.e. asfor example described in the International application WO 03/050531(Lasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different Nanobodiesof the invention), i.e. to provide an analog (as defined herein) and/orto provide further parts or fragments (as defined herein) of a Nanobodyof the invention. It is for example also possible to combine one or moreparts or fragments of a Nanobody of the invention with one or more partsor fragments of a human V_(H) domain.

According to one preferred aspect, the parts or fragments have a degreeof sequence identity of at least 50%, preferably at least 60%, morepreferably at least 70%, even more preferably at least 80%, such as atleast 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs705 to 788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to764, 772 to 773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773or 778 (see Table A-1).

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized Nanobody of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized Nanobody of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.g.enzymatical) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or animmunoglobulin sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to, thefluorescent labels, phosphorescent labels, chemiluminescent labels,bioluminescent labels, radio-isotopes, metals, metal chelates, metalliccations, chromophores and enzymes, such as those mentioned on page 109of WO 08/020079. Other suitable labels will be clear to the skilledperson, and for example include moieties that can be detected using NMRor ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to on channels suchas e.g. P2X7 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein for the Nanobodiesof the invention.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least oneNanobody of the invention. By “essentially consist of” is meant that theimmunoglobulin sequence of the polypeptide of the invention either isexactly the same as the immunoglobulin sequence of a Nanobody of theinvention or corresponds to the immunoglobulin sequence of a Nanobody ofthe invention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe immunoglobulin sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   -   can comprise an N-terminal Met residue, for example as result of        expression in a heterologous host cell or host organism.    -   may form a signal sequence or leader sequence that directs        secretion of the Nanobody from a host cell upon synthesis.        Suitable secretory leader peptides will be clear to the skilled        person, and may be as further described herein. Usually, such a        leader sequence will be linked to the N-terminus of the        Nanobody, although the invention in its broadest sense is not        limited thereto;    -   may form a sequence or signal that allows the Nanobody to be        directed towards and/or to penetrate or enter into specific        organs, tissues, cells, or parts or compartments of cells,        and/or that allows the Nanobody to penetrate or cross a        biological barrier such as a cell membrane, a cell layer such as        a layer of epithelial cells, a tumor including solid tumors, or        the blood-brain-barrier. Examples of such immunoglobulin        sequences will be clear to the skilled person and include those        mentioned in paragraph c) on page 112 of WO 08/020079.    -   may form a “tag”, for example an immunoglobulin sequence or        residue that allows or facilitates the purification of the        Nanobody, for example using affinity techniques directed against        said sequence or residue. Thereafter, said sequence or residue        may be removed (e.g. by chemical or enzymatical cleavage) to        provide the Nanobody sequence (for this purpose, the tag may        optionally be linked to the Nanobody sequence via a cleavable        linker sequence or contain a cleavable motif). Some preferred,        but non-limiting examples of such residues are multiple        histidine residues, glutathione residues and a myc-tag (see for        example SEQ ID NO:31 of WO 06/12282).    -   may be one or more amino acid residues that have been        functionalized and/or that can serve as a site for attachment of        functional groups. Suitable amino acid residues and functional        groups will be clear to the skilled person and include, but are        not limited to, the amino acid residues and functional groups        mentioned herein for the derivatives of the Nanobodies of the        invention.        DNA Vaccination in Camelids

DNA vaccination has been widely used for inducing immune responses inexperimental animals, such as rats, mice and rabbits (see e.g. Tang etal., Nature, 1992; Nagata et al., J. Immunol. Methods, 2003; Bins etal., Nature Med. 2005; Bins et al, J. Immunol., 2007; Kilpatrick et al.,Hybridoma, 1998). However, DNA vaccination of larger animals has beenconsidered difficult, if not impossible. In particular, reports on DNAvaccination of camelids are scarce. The report by Koch Nolte et al.,2007, describes a lengthy and cumbersome procedure requiring no lessthan 8 rounds of DNA vaccination, at 6-12 week intervals, in combinationwith a further two rounds of protein boost. It is one objective of thepresent invention to provide an improved method for DNA vaccination oflarge animals, in particular camelids.

a) Generation of Immunoglobulin Sequences without Protein Boost

In a particular embodiment, the present invention relates to a methodfor generating immunoglobulin sequences in a non-human animal,specifically in a camelid, more specifically a llama, by geneticvaccination, without protein boost.

It has previously been demonstrated in non-camelids, e.g. in mice, thatpolyclonal serum antibody responses can be obtained after DNAvaccination, without boost with a protein antigen (e.g. Bins et al.,Nature Medicine, 2005; Bins et al., J. Immunol. 2007). However, it isknown that such polyclonal antibody responses are not sufficient toefficiently generate antigen specific monoclonal antibodies (Nagata etal., 2003). The authors report a specific hybridoma yield that was“almost negligible” (0.0-1.3/10E8 spleen cells) in mice in the absenceof a protein boost.

It is generally known that genetic vaccination of larger animals, inparticular camelids, is far less efficient in eliciting an immuneresponse as compared to mice. The production of immunoglobulin sequencesby use of genetic vaccination in camelids has therefore been consideredimpossible.

Koch-Nolte et al. 2007 describe the immunization of a llama with aDNA-prime protein boost strategy for obtaining single domain antibodiesagainst ecto-ADP-ribosyltransferase ART2.2. This procedure, however, wascharacterized by low efficiency and required extensive boosting. Morespecifically, a llama received four intradermal gene gun immunizationswith an expression vector encoding ART2.2 at 6 to 12 week intervals (12shots of 1 μg DNA/mg gold at a pressure setting of 300 psi). Foursubsequent boost DNA immunizations and a further two protein boosts wererequired using recombinant ART2.2, in order to obtain a satisfactoryimmune response. Overall, this procedure extends over several monthsprior to achieving a suitable immune response in the animal, andnecessitates multiple protein boosts.

In view of this prior art teaching, it is a surprising finding of thepresent invention that by using the methods as described herein, asuitable immune response can be achieved in camelids, in particularllamas, without a protein boost. In other words, genetic vaccinationalone can suffice to induce an immune response that is adequate for thegeneration of immunoglobulin sequences by subsequent screening. Inparticular it has surprisingly been found that even if the serumantibody response is low as compared to conventional immunizationstrategies, the DNA vaccination alone suffices to achieve good“hit-rates”, i.e. to obtain antigen specific immunoglobulin sequences atan acceptable frequency.

b) Generation of Antibody Responses to Cell Associated Antigens

As outlined in the introductory section, the art does not provide foradequate means to generate antibody responses to antigens which are cellassociated as defined herein, in particular to transmembrane antigens.

The present invention is based on the surprising finding that by geneticvaccination of suitable non-human animals, in particular camelids,preferably llamas, antibody responses can be generated, which arecharacterized by an improved breadth of the repertoire as well as animproved specificity as compared to prior art attempts.

Cell-associated antigens, more specifically those with single ormultiple transmembrane domains, are difficult to purify in their naturalconformation. In order to obtain immunoglobulin sequences, includingNanobodies, against native epitopes, it is crucial to administer thetarget antigen to the llama in its native conformation. For thesecell-associated antigens, immunization with whole cells functionallyexpressing the antigen is the preferred strategy (as was done e.g. in WO05/044858; WO 07/042289; U.S. 61/004,332). The main disadvantage ofwhole cell immunizations is the fact that many other antigens (cellsurface markers) are also presented to the immune system of the animal,which results in a highly diluted target specific immune response. Toincrease the specificity, elaborate and technically complex prime-booststrategies have to be devised. To the extent any of these steps includesthe use of denatured protein or peptides, the resulting antibodyresponse will be biased to the disadvantage of conformational epitopes.Therefore, the breadth of the obtainable spectrum of immunoglobulinsequences will be limited. Moreover, solely cell-based approaches, i.e.approaches which use cells expressing the target antigen forimmunization, will be characterized by poor specificity of theimmunoglobulin sequences, rendering an efficient isolation ofimmunoglobulin sequences of interest impossible.

Koch-Nolte et al. 2007 describe the immunization of one llama with aDNA-prime protein boost strategy for obtaining single domain antibodiesagainst ecto-ADP-ribosyltransferase ART2.2.

Ecto-ADP-ribosyltransferase ART2.2 is a Gpi anchored protein,characterized by a membrane insertion via a lipid tail but without atransmembrane domain. For this protein, correctly folded purifiedprotein could be prepared for the boost. Therefore a boost could be donewith the purified protein. As indicated above, purified protein ofcell-associated antigens that are anchored within, or located in themembrane can not be obtained in a purified form in their naturalconformation. Any purified preparations will have lost the membranedependent conformational epitopes. And therefore, a boost with purifiedprotein from these cell-associated antigens is not possible.

In the context of transmembrane proteins, and in particular proteinswith multiple transmembrane domains, conformational epitopes, and inparticular membrane-dependent conformational epitopes are of particularinterest as targets for immunoglobulin sequences. For example, the poreof an ion channel represents a target of primary therapeutic importance.However, by use of conventional approaches, it is nearly impossible togenerate immunoglobulin sequences that recognize such a target. To staywith the example, the pore region of an ion channel is formed bymultiple membrane spanning domains, and possibly even multiple subunitsof the channel. It is near impossible to provide peptides for thegeneration of immunoglobulin sequences binding to this pore region.Moreover, because the protein will only exhibit its natural conformationin the membrane environment, purified ion channel protein cannot be usedfor immunization.

The present invention provides for the generation of immunoglobulinsequences to such kind of conformational epitope, and excludes the needfor boost with purified protein.

In the invention it is envisaged that after genetic vaccination (whichprovides for the necessary specificity of the immune response), ananimal can be boosted with e.g. cells expressing the protein in itsnatural conformation, i.e. embedded/anchored in the membrane. Eventhough these cells will express a multitude of antigens, theimmunological recall response will only occur for the antigen that hasbeen delivered by genetic vaccination. Thus, priming the animal withgenetic vaccination allows a boost with protein in its naturalconformation, even if the protein is non-purified, e.g. in the contextof a cell expressing the protein, but nevertheless obtaining a highlyspecific repertoire of immunoglobulin sequences.

In a preferred embodiment, the invention relates to the generation ofimmunoglobulin sequences in camelids, in particular llama. The antibodyresponse of these animals is characterized by the existence ofimmunoglobulin sequences that can extend into, and specifically bind togrooves or crevices on a target antigen. This is of particularimportance and benefit in the case of conformational epitopes of cellassociated antigens

Thus, in one specific embodiment the present invention relates to theuse of genetic vaccination of camelids, to raise immunoglobulinsequences against conformational epitopes of cell-associated antigens,in particular antigens exhibiting one or multiple membrane spanningdomains.

The general principles of the present invention as set forth above willnow be exemplified by reference to specific preferred aspects,experiments and claims. However, the invention is not to be understoodas being limited thereto.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

Preferred Aspects:

-   Aspect A-1: An immunoglobulin sequence that is directed against    and/or that can specifically bind to ion channels such as e.g. P2X7.-   Aspect A-2: An immunoglobulin sequence according to aspect A-1, that    is in essentially isolated form.-   Aspect A-3: An immunoglobulin sequence according to aspect A-1 or    A-2, for administration to a subject, wherein said immunoglobulin    sequence does not naturally occur in said subject.-   Aspect A-4: An immunoglobulin sequence that can specifically bind to    ion channels such as e.g. P2X7 with a dissociation constant (K_(D))    of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.    Such an immunoglobulin sequence may in particular be an    immunoglobulin sequence according to any of the preceding aspects.-   Aspect A-5: An immunoglobulin sequence that can specifically bind to    ion channels such as e.g. P2X7 with a rate of association    (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably    between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴    M⁻¹s⁻¹ and 10⁷M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.    Such an immunoglobulin sequence may in particular be an    immunoglobulin sequence according to any of the preceding aspects.-   Aspect A-6: An immunoglobulin sequence that can specifically bind to    ion channels such as e.g. P2X7 with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹, preferably between 10⁻² s¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹. Such an immunoglobulin sequence may    in particular be an immunoglobulin sequence according to any of the    preceding aspects.-   Aspect A-7: An immunoglobulin sequence that can specifically bind to    ion channels such as e.g. P2X7 with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Such an immunoglobulin sequence may in    particular be an immunoglobulin sequence according to any of the    preceding aspects.-   Aspect A-8: An immunoglobulin sequence according to any of the    preceding aspects, that is a naturally occurring immunoglobulin    sequence (from any suitable species) or a synthetic or    semi-synthetic immunoglobulin sequence.-   Aspect A-9: An immunoglobulin sequence according to any of the    preceding aspects, that comprises an immunoglobulin fold or that    under suitable conditions is capable of forming an immunoglobulin    fold.-   Aspect A-10: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of 4 framework regions    (FR1 to FR4 respectively) and 3 complementarity determining regions    (CDR1 to CDR3 respectively).-   Aspect A-11: An immunoglobulin sequence according to any of the    preceding aspects, that is an immunoglobulin sequence.-   Aspect A-12: An immunoglobulin sequence according to any of the    preceding aspects, that is a naturally occurring immunoglobulin    sequence (from any suitable species) or a synthetic or    semi-synthetic immunoglobulin sequence.-   Aspect A-13: An immunoglobulin sequence according to any of the    preceding aspects that is a humanized immunoglobulin sequence, a    camelized immunoglobulin sequence or an immunoglobulin sequence that    has been obtained by techniques such as affinity maturation.-   Aspect A-14: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of a light chain    variable domain sequence (e.g. a VL-sequence); or of a heavy chain    variable domain sequence (e.g. a VH-sequence).-   Aspect A-15: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of a heavy chain    variable domain sequence that is derived from a conventional    four-chain antibody or that essentially consist of a heavy chain    variable domain sequence that is derived from heavy chain antibody.-   Aspect A-16: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of a domain antibody    (or an immunoglobulin sequence that is suitable for use as a domain    antibody), of a single domain antibody (or an immunoglobulin    sequence that is suitable for use as a single domain antibody), of a    “dAb” (or an immunoglobulin sequence that is suitable for use as a    dAb) or of a Nanobody (including but not limited to a VHH sequence).-   Aspect A-17: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of a Nanobody.-   Aspect A-18: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        An immunoglobulin sequences of SEQ ID NO's: 1 to 22, in which        for the purposes of determining the degree of amino acid        identity, the amino acid residues that form the CDR sequences        are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect A-19: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of a polypeptide that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin sequences of SEQ ID NO's: 789 to 791, in which        for the purposes of determining the degree of amino acid        identity, the amino acid residues that form the CDR sequences        are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect A-20: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin sequences of SEQ ID NO's: 705 to 788, more        preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772        to 773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773        or 778, in which for the purposes of determining the degree of        amino acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect A-21: An immunoglobulin sequence according to any of the    preceding aspects, that essentially consists of a humanized    Nanobody.-   Aspect A-22: An immunoglobulin sequence according to any of the    preceding aspects, that in addition to the at least one binding site    for binding against ion channels such as e.g. P2X7, contains one or    more further binding sites for binding against other antigens,    proteins or targets.    CDR-Based Aspects-   Aspect B-1: An immunoglobulin sequence that is directed against    and/or that can specifically bind ion channels such as e.g. P2X7,    and that comprises one or more stretches of amino acid residues    chosen from the group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   i) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   or any suitable combination thereof.

Such an immunoglobulin sequence may in particular be an immunoglobulinsequence according to any of the aspects A-1 to A-22.

-   Aspect B-2: An immunoglobulin sequence according to aspect B-1, in    which at least one of said stretches of amino acid residues forms    part of the antigen binding site for binding against ion channels    such as e.g. P2X7.-   Aspect B-3: An immunoglobulin sequence that is directed against    and/or that can specifically bind ion channels such as e.g. P2X7 and    that comprises two or more stretches of amino acid residues chosen    from the group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   i) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the immunoglobulin sequences according to        a), b) or c), the second stretch of amino acid residues        corresponds to one of the immunoglobulin sequences according to        d), e), f), g), h) or i); (ii) when the first stretch of amino        acid residues corresponds to one of the immunoglobulin sequences        according to d), e) or f), the second stretch of amino acid        residues corresponds to one of the immunoglobulin sequences        according to a), b), c), g), h) or i); or (iii) when the first        stretch of amino acid residues corresponds to one of the        immunoglobulin sequences according to g), h) or i), the second        stretch of amino acid residues corresponds to one of the        immunoglobulin sequences according to a), b), c), d), e) or f).

Such an immunoglobulin sequence may in particular be an immunoglobulinsequence according to any of the aspects A-1 to A-22, B-1 or B-2.

-   Aspect B-4: An immunoglobulin sequence according to aspect B-3, in    which the at least two stretches of amino acid residues forms part    of the antigen binding site for binding against ion channels such as    e.g. P2X7.-   Aspect B-5: An immunoglobulin sequence that is directed against    and/or that can specifically bind on channels such as e.g. P2X7 and    that comprises three or more stretches of amino acid residues, in    which the first stretch of amino acid residues is chosen from the    group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   the second stretch of amino acid residues is chosen from the        group consisting of:    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   and the third stretch of amino acid residues is chosen from the        group consisting of:    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   i) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617.

Such an immunoglobulin sequence may in particular be an immunoglobulinsequence according to any of the aspects A-1 to A-22 and/or B-1 to B-4.

-   Aspect B-6: An immunoglobulin sequence according to aspect B-5, in    which the at least three stretches of amino acid residues forms part    of the antigen binding site for binding against ion channels such as    e.g. P2X7.-   Aspect B-7: An immunoglobulin sequence that is directed against    and/or that can specifically bind ion channels such as e.g. P2X7 in    which the CDR sequences of said immunoglobulin sequence have at    least 70% amino acid identity, preferably at least 80% amino acid    identity, more preferably at least 90% amino acid identity, such as    95% amino acid identity or more or even essentially 100% amino acid    identity with the CDR sequences of at least one of the    immunoglobulin sequences of SEQ ID NO's: 705 to 788, more preferably    SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or    778 to 780, more preferred SEQ ID NO's 732, 773 or 778. Such an    immunoglobulin sequence may in particular be an immunoglobulin    sequence according to any of the aspects A-1 to A-22 and/or B-1 to    B-6.-   Aspect C-1: An immunoglobulin sequence that is directed against ion    channels such as e.g. P2X7 and that cross-blocks the binding of at    least one of the immunoglobulin sequences of SEQ ID NO's: 705 to    788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764,    772 to 773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773    or 778 to ion channels such as e.g. P2X7. Such an immunoglobulin    sequence may in particular be an immunoglobulin sequence according    to any of the aspects A-1 to A-22 and/or according to aspects B-1 to    B-7. Also, preferably, such an immunoglobulin sequence is able to    specifically bind to ion channels such as e.g. P2X7.-   Aspect C-2: An immunoglobulin sequence that is directed against ion    channels such as e.g. P2X7 and that is cross-blocked from binding to    ion channels such as e.g. P2X7 by at least one of the immunoglobulin    sequences of SEQ ID NO's: 705 to 788, more preferably SEQ ID NO's    726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or 778 to 780,    more preferred SEQ ID NO's 732, 773 or 778. Such an immunoglobulin    sequence may in particular be an immunoglobulin sequence according    to any of the aspects A-1 to A-22 and/or according to aspects B-1 to    B-7. Also, preferably, such an immunoglobulin sequence is able to    specifically bind to ion channels such as e.g. P2X7.-   Aspect C-3: An immunoglobulin sequence according to any of aspects    C-1 or C-2, wherein the ability of said immunoglobulin sequence to    cross-block or to be cross-blocked is detected in a Biacore assay.-   Aspect C-4: An immunoglobulin sequence according to any of aspects    C-1 to C-3 wherein the ability of said immunoglobulin sequence to    cross-block or to be cross-blocked is detected in an ELISA assay.-   Aspect D-1: An immunoglobulin sequence according to any of aspects    B-1 to B-7 or C-1 to C-7, that is in essentially isolated form.-   Aspect D-2: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, and/or D1 for administration to a subject,    wherein said immunoglobulin sequence does not naturally occur in    said subject.-   Aspect D-3: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, and/or D1 to D-2 that can specifically bind    to ion channels such as e.g. P2X7 with a dissociation constant    (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to    10⁻¹² moles/liter or less and more preferably 10⁻⁵ to 10⁻¹²    moles/liter.-   Aspect D-4: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, and/or D-1 to D-3 that can specifically bind    to ion channels such as e.g. P2X7 with a rate of association    (k_(on)-rate) of between 102 M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably    between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect D-5: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, and/or D-1 to D-4 that can specifically bind    to ion channels such as e.g. P2X7 with a rate of dissociation    (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻²    s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹,    such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect D-6: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, and/or D-1 to D-5 that can specifically bind    to ion channels such as e.g. P2X7 with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM.

The immunoglobulin sequences according to aspects D-1 to D-6 may inparticular be an immunoglobulin sequence according to any of the aspectsA-1 to A-22.

-   Aspect E-1: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7 and/or D1 to D-6, that is a naturally    occurring immunoglobulin sequence (from any suitable species) or a    synthetic or semi-synthetic immunoglobulin sequence.-   Aspect E-2: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 that comprises an    immunoglobulin fold or that under suitable conditions is capable of    forming an immunoglobulin fold.-   Aspect E-3: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or D-1 or D-2, that is an    immunoglobulin sequence.-   Aspect E-4: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-3, that is a    naturally occurring immunoglobulin sequence (from any suitable    species) or a synthetic or semi-synthetic immunoglobulin sequence.-   Aspect E-5: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-4 that is a    humanized immunoglobulin sequence, a camelized immunoglobulin    sequence or an immunoglobulin sequence that has been obtained by    techniques such as affinity maturation.-   Aspect E-6: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-5 that    essentially consists of a light chain variable domain sequence (e.g.    a V_(L)-sequence); or of a heavy chain variable domain sequence    (e.g. a V_(H)-sequence).-   Aspect E-7: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-6, that    essentially consists of a heavy chain variable domain sequence that    is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.-   Aspect E-8: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-7, that    essentially consists of a domain antibody (or an immunoglobulin    sequence that is suitable for use as a domain antibody), of a single    domain antibody (or an immunoglobulin sequence that is suitable for    use as a single domain antibody), of a “dAb” (or an immunoglobulin    sequence that is suitable for use as a dAb) or of a Nanobody    (including but not limited to a V_(HH) sequence).-   Aspect E-9: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E1 to E-8 that essentially    consists of a Nanobody.-   Aspect E-10: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-9 that    essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin sequences of SEQ ID NO's: 1 to 22, in which for        the purposes of determining the degree of amino acid identity,        the amino acid residues that form the CDR sequences are        disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect E-11: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E1 to E-10, that    essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        An immunoglobulin sequences of SEQ ID NO's: 705 to 788, more        preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772        to 773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773        or 778, in which for the purposes of determining the degree of        amino acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect E-12: An immunoglobulin sequence according to any of aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-11 that    essentially consists of a humanized Nanobody.-   Aspect E-13: An immunoglobulin sequence according to any of the    aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E1 to E-11, that    in addition to the at least one binding site for binding formed by    the CDR sequences, contains one or more further binding sites for    binding against other antigens, proteins or targets.

The immunoglobulin sequences according to aspects E-1 to E-13 may inparticular be an immunoglobulin sequence according to any of the aspectsA-1 to A-22.

Framework+CDR's Aspects

-   Aspect F-1: An immunoglobulin sequence that essentially consists of    4 framework regions (FR1 to FR4, respectively) and 3 complementarity    determining regions (CDR1 to CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   i) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617.

Such an immunoglobulin sequence is preferably directed against ionchannels such as e.g. P2X7 and/or an immunoglobulin sequence that canspecifically bind to ion channels such as e.g. P2X7. Also, such animmunoglobulin sequence is preferably an immunoglobulin sequenceaccording to any of the aspects A-1 to A-22, C-1 to C-7, D1 to D-6and/or E1 to E-13.

-   Aspect F-2: An immunoglobulin sequence that essentially consists of    4 framework regions (FR1 to FR4, respectively) and 3 complementarity    determining regions (CDR1 to CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   and    -   CDR2 is chosen from the group consisting of:    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   and    -   CDR3 is chosen from the group consisting of:    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   i) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617.

Such an immunoglobulin sequence is preferably directed against ionchannels such as e.g. P2X7 and/or an immunoglobulin sequence that canspecifically bind to ion channels such as e.g. P2X7. Also, such animmunoglobulin sequence is preferably an immunoglobulin sequenceaccording to any of the aspects A-1 to A-22, C-1 to C-7, D1 to D-6and/or E1 to E-13.

-   -   Aspect F-3: An immunoglobulin sequence according to any of        aspects F-1 and F-2, in which the CDR sequences of said        immunoglobulin sequence have at least 70% amino acid identity,        preferably at least 80% amino acid identity, more preferably at        least 90% amino acid identity, such as 95% amino acid identity        or more or even essentially 100% amino acid identity with the        CDR sequences of at least one of the immunoglobulin sequences of        SEQ ID NO's: 705 to 788, more preferably SEQ ID NO's 726 to 750,        753 to 758, 762 to 764, 772 to 773, 775, or 778 to 780, more        preferred SEQ ID NO's 732, 773 or 778.

Such an immunoglobulin sequence is preferably directed against ionchannels such as e.g. P2X7 and/or an immunoglobulin sequence that canspecifically bind to ion channels such as e.g. P2X7. Also, such animmunoglobulin sequence is preferably an immunoglobulin sequenceaccording to any of the aspects A-1 to A-22, C-1 to C-7, D1 to D-6and/or E-1 to E-13.

-   Aspect F-4: An immunoglobulin sequence according to any of aspects    F-1 to F-3 that is directed against ion channels such as e.g. P2X7    and that cross-blocks the binding of at least one of the    immunoglobulin sequences according to any of aspects the    immunoglobulin sequences of SEQ ID NO's: 705 to 788, more preferably    SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or    778 to 780, more preferred SEQ ID NO's 732, 773 or 778.-   Aspect F-5: An immunoglobulin sequence according to any of aspects    F-1 to F-3 that is directed against ion channels such as e.g. P2X7    and that is cross-blocked from binding to ion channels such as e.g.    P2X7 by at least one of the immunoglobulin sequences of SEQ ID NO's:    705 to 788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762    to 764, 772 to 773, 775, or 778 to 780, more preferred SEQ ID NO's    732, 773 or 778.-   Aspect F-6: Immunoglobulin sequence according to any of aspects F-4    or F-5 wherein the ability of said immunoglobulin sequence to    cross-block or to be cross-blocked is detected in a Biacore assay.-   Aspect F-7: Immunoglobulin sequence according to any of aspects F4    or F-5 wherein the ability of said immunoglobulin sequence to    cross-block or to be cross-blocked is detected in an ELISA assay.-   Aspect F-8: An immunoglobulin sequence according to any of aspects    F-1 to F-7, that is in essentially isolated form.-   Aspect F-9: An immunoglobulin sequence according to any of aspects    F-1 to F-8, for administration to a subject, wherein said an    immunoglobulin sequence does not naturally occur in said subject.-   Aspect F-10: An immunoglobulin sequence according to any of aspects    F-1 to F-9, that can specifically bind to ion channels such as e.g.    P2X7 with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²    moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or    less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.-   Aspect F-11: An immunoglobulin sequence according to any of aspects    F-1 to F-10, that can specifically bind to ion channels such as e.g.    P2X7 with a rate of association (k_(on)-rate) of between 10² M⁻¹s⁻¹    to about 10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,    more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between    10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect F-12: An immunoglobulin sequence according to any of aspects    F-1 to F-11, that can specifically bind to ion channels such as e.g.    P2X7 with a rate of dissociation (k_(off) rate) between 1 s⁻¹ and    10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s¹, more preferably    between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶    s⁻¹.-   Aspect F-13: An immunoglobulin sequence according to any of aspects    F-1 to F-12, that can specifically bind to ion channels such as e.g.    P2X7 with an affinity less than 500 nM, preferably less than 200 nM,    more preferably less than 10 nM, such as less than 500 μM.-   Aspect F-14: An immunoglobulin sequence according to any of aspects    F-1 to F-13, that is a naturally occurring immunoglobulin sequence    (from any suitable species) or a synthetic or semi-synthetic    immunoglobulin sequence.-   Aspect F-15: An immunoglobulin sequence according to any of aspects    F-1 to F-14, that comprises an immunoglobulin fold or that under    suitable conditions is capable of forming an immunoglobulin fold.-   Aspect F-16: An immunoglobulin sequence according to any of aspects    F-1 to F-15, that is an immunoglobulin sequence.-   Aspect F-17: An immunoglobulin sequence according to any of aspects    F-1 to F-16, that is a naturally occurring immunoglobulin sequence    (from any suitable species) or a synthetic or semi-synthetic    immunoglobulin sequence.-   Aspect F-18: An immunoglobulin sequence according to any of aspects    F-1 to F-17, that is a humanized immunoglobulin sequence, a    camelized immunoglobulin sequence or an immunoglobulin sequence that    has been obtained by techniques such as affinity maturation.-   Aspect F-19: An immunoglobulin sequence according to any of aspects    F-1 to F-19, that essentially consists of a light chain variable    domain sequence (e.g. a V_(L)-sequence); or of a heavy chain    variable domain sequence (e.g. a V_(H)-sequence).-   Aspect F-20: An immunoglobulin sequence according to any of aspects    F-1 to F-19, that essentially consists of a heavy chain variable    domain sequence that is derived from a conventional four-chain    antibody or that essentially consist of a heavy chain variable    domain sequence that is derived from heavy chain antibody.-   Aspect F-21: An immunoglobulin sequence according to any of aspects    F-1 to F-20, that essentially consists of a domain antibody (or an    immunoglobulin sequence that is suitable for use as a domain    antibody), of a single domain antibody (or an immunoglobulin    sequence that is suitable for use as a single domain antibody), of a    “dAb” (or an immunoglobulin sequence that is suitable for use as a    dAb) or of a Nanobody (including but not limited to a V_(HH)    sequence).-   Aspect F-22: An immunoglobulin sequence according to any of aspects    F-1 to F-21, that essentially consists of a Nanobody.-   Aspect F-23: An immunoglobulin sequence according to any of aspects    F-1 to F-22, that essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin sequences of SEQ ID NO's: 1 to 22, in which for        the purposes of determining the degree of amino acid identity,        the amino acid residues that form the CDR sequences are        disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect F-24: An immunoglobulin sequence according to any of aspects    F-1 to F-23, that essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin sequences of SEQ ID NO's: 705 to 788, more        preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772        to 773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773        or 778, in which for the purposes of determining the degree of        amino acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect F-25: An immunoglobulin sequence according to any of aspects    F-1 to F-24, that essentially consists of a humanized Nanobody.-   Aspect G-1: An immunoglobulin sequence according to any of the    preceding aspects, that in addition to the at least one binding site    for binding formed by the CDR sequences, contains one or more    further binding sites for binding against another antigen, protein    or target.-   Aspect H-1: Nanobody that is directed against and/or that can    specifically bind to ion channels such as e.g. P2X7.-   Aspect H-2: Nanobody according to aspect H-1, that is in essentially    isolated form.-   Aspect H-3: Nanobody according to any of aspects H-1 to H-2, that    can specifically bind to ion channels such as e.g. P2X7 with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less,    and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably    10⁻⁸ to 10⁻¹² moles/liter.-   Aspect H-4: Nanobody according to any of aspects H-1 to H-3, that    can specifically bind to ion channels such as e.g. P2X7 with a rate    of association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷    M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more    preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect H-5: Nanobody according to any of aspects H-1 to H-4, that    can specifically bind to ion channels such as e.g. P2X7 with a rate    of dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably    between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁶ s⁻¹ and    10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect H-6: Nanobody according to any of aspects H-1 to H-5, that    can specifically bind to on channels such as e.g. P2X7 with an    affinity less than 500 nM, preferably less than 200 nM, more    preferably less than 10 nM, such as less than 500 μM.-   Aspect H-7: Nanobody according to any of aspects H-1 to H-6, that is    a naturally occurring Nanobody (from any suitable species) or a    synthetic or semi-synthetic Nanobody.-   Aspect H-8: Nanobody according to any of aspects to H-1 to H-7, that    is a V_(HH) sequence, a partially humanized V_(HH) sequence, a fully    humanized V_(HH) sequence, a camelized heavy chain variable domain    or a Nanobody that has been obtained by techniques such as affinity    maturation.-   Aspect H-9: Nanobody according to any of aspects H-1 to H-8, that    -   i) has at least 80% amino acid identity with at least one of the        An immunoglobulin sequences of SEQ ID NO's: 1 to 22, in which        for the purposes of determining the degree of amino acid        identity, the amino acid residues that form the CDR sequences        are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect H-10: Nanobody according to any of aspects H-1 to H-9, that    -   i) has at least 80% amino acid identity with at least one of the        An immunoglobulin sequences of SEQ ID NO's: 705 to 788, more        preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772        to 773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773        or 778, in which for the purposes of determining the degree of        amino acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect H-11: Nanobody according to any of aspects H-1 to H-10, in    which:    -   CDR1 is chosen from the group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   i) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617.-   Aspect H-12: Nanobody according to any of aspects H-1 to H-11, in    which:    -   CDR1 is chosen from the group consisting of:    -   a) the immunoglobulin sequences of SEQ ID NO's: 208 to 289;    -   b) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   c) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 208 to 289;    -   and    -   CDR2 is chosen from the group consisting of:    -   d) the immunoglobulin sequences of SEQ ID NO's: 372 to 453;    -   e) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   f) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 372 to 453;    -   and    -   CDR3 is chosen from the group consisting of:    -   g) the immunoglobulin sequences of SEQ ID NO's: 536 to 617;    -   h) immunoglobulin sequences that have at least 80% amino acid        identity with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617;    -   i) immunoglobulin sequences that have 3, 2, or 1 amino acid        difference with at least one of the immunoglobulin sequences of        SEQ ID NO's: 536 to 617.-   Aspect H-13: Nanobody according to any of aspects H-1 to H-12, in    which the CDR sequences have at least 70% amino acid identity,    preferably at least 80% amino acid identity, more preferably at    least 90% amino acid identity, such as 95% amino acid identity or    more or even essentially 100% amino acid identity with the CDR    sequences of at least one of the immunoglobulin sequences of SEQ ID    NO's: 705 to 788, more preferably SEQ ID NO's 726 to 750, 753 to    758, 762 to 764, 772 to 773, 775; or 778 to 780, more preferred SEQ    ID NO's 732, 773 or 778.-   Aspect H-14: Nanobody according to any of aspects H-1 to H-13, which    is a partially humanized Nanobody.-   Aspect H-15: Nanobody according to any of aspects H-1 to H-14, which    is a fully humanized Nanobody.-   Aspect H-16: Nanobody according to any of aspects H-1 to H-15, that    is chosen from the group consisting of SEQ ID NO's: 705 to 788, more    preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to    773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773 or 778    or from the group consisting of from immunoglobulin sequences that    have more than 80%, preferably more than 90%, more preferably more    than 95%, such as 99% or more sequence identity (as defined herein)    with at least one of the immunoglobulin sequences of SEQ ID NO's:    705 to 788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762    to 764, 772 to 773, 775, or 778 to 780, more preferred SEQ ID NO's    732, 773 or 778.-   Aspect H-17: Nanobody according to any of aspects H-1 to H-16, which    is a humanized Nanobody.-   Aspect H-18: Nanobody according to any of aspects H-1 to H-17, that    is chosen from the group consisting of SEQ ID NO's: 705 to 788, more    preferably SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to    773, 775, or 778 to 780, more preferred SEQ ID NO's 732, 773.-   Aspect H-19: Nanobody directed against ion channels such as e.g.    P2X7 that cross-blocks the binding of at least one of the    immunoglobulin sequences of SEQ ID NO's: 705 to 788, more preferably    SEQ ID NO's 726 to 750, 753 to 758, 762 to 764, 772 to 773, 775, or    778 to 780, more preferred SEQ ID NO's 732, 773 or 778 to ion    channels such as e.g. P2X7.-   Aspect H-20: Nanobody directed against ion channels such as e.g.    P2X7 that is cross-blocked from binding to ion channels such as e.g.    P2X7 by at least one of the immunoglobulin sequences of SEQ ID NO's:    705 to 788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762    to 764, 772 to 773, 775, or 778 to 780, more preferred SEQ ID NO's    732, 773 or 778.-   Aspect H-21: Nanobody according to any of aspects H-19 or H-20    wherein the ability of said Nanobody to cross-block or to be    cross-blocked is detected in a Biacore assay.-   Aspect H-22: Nanobody according to any of aspects H-19 to H-21    wherein the ability of said Nanobody to cross-block or to be    cross-blocked is detected in an ELISA assay.    Polypeptides.-   Aspect K-1: Polypeptide that comprises or essentially consists of    one or more immunoglobulin sequences according to any of aspects A-1    to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1 and/or one or more Nanobodies according to any of    aspects H-1 to H-22, and optionally further comprises one or more    peptidic linkers.-   Aspect K-2: Polypeptide according to aspect K-1, in which said one    or more binding units are immunoglobulin sequences.-   Aspect K-3: Polypeptide according to any of aspects K-1 or K-2, in    which said one or more other groups, residues, moieties or binding    units are chosen from the group consisting of domain antibodies,    immunoglobulin sequences that are suitable for use as a domain    antibody, single domain antibodies, immunoglobulin sequences that    are suitable for use as a single domain antibody, “dAb”'s,    immunoglobulin sequences that are suitable for use as a dAb, or    Nanobodies.-   Aspect K-4: Polypeptide according to any of aspects K-1 to K-3, in    which said one or more immunoglobulin sequences of the invention are    immunoglobulin sequences.-   Aspect K-5: Polypeptide according to any of aspects K-1 to K-4, in    which said one or more immunoglobulin sequences of the invention are    chosen from the group consisting of domain antibodies,    immunoglobulin sequences that are suitable for use as a domain    antibody, single domain antibodies, immunoglobulin sequences that    are suitable for use as a single domain antibody, “dAb”'s,    immunoglobulin sequences that are suitable for use as a dAb, or    Nanobodies.-   Aspect K-6: Polypeptide according to any of aspects K-1 to K-5, that    comprises or essentially consists of one or more Nanobodies    according to any of aspects H-1 to H-22 and in which said one or    more other binding units are Nanobodies.-   Aspect K-7: Polypeptide according to any of aspects K-1 to K-6,    wherein at least one binding unit is a multivalent construct.-   Aspect K-8: Polypeptide according to any of aspects K-1 to K-8,    wherein at least one binding unit is a multiparatopic construct.-   Aspect K-9: Polypeptide according to any of aspects K-1 to K-8,    wherein at least one binding unit is a multispecific construct.-   Aspect K-10: Polypeptide according to any of aspects K-1 to K-9,    which has an increased half-life, compared to the corresponding    immunoglobulin sequence according to any of aspects A-1 to A-22, B-1    to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1 per    se or Nanobody according to any of aspects H-1 to H-22 per se,    respectively.-   Aspect K-11: Polypeptide according to aspect K-10, in which said one    or more other binding units provide the polypeptide with increased    half-life, compared to the corresponding immunoglobulin sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se or Nanobody according    to any of aspects H-1 to H-22 per se, respectively.-   Aspect K-12: Polypeptide according to aspect K-10 or K-11, in which    said one or more other binding units that provide the polypeptide    with increased half-life is chosen from the group consisting of    serum proteins or fragments thereof, binding units that can bind to    serum proteins, an Fc portion, and small proteins or peptides that    can bind to serum proteins.-   Aspect K-13: Polypeptide according to any of aspects K-10 to K-12,    in which said one or more other binding units that provide the    polypeptide with increased half-life is chosen from the group    consisting of human serum albumin or fragments thereof.-   Aspect K-14: Polypeptide according to any of aspect K-10 to K-13, in    which said one or more other binding units that provides the    polypeptide with increased half-life are chosen from the group    consisting of binding units that can bind to serum albumin (such as    human serum albumin) or a serum immunoglobulin (such as IgG).-   Aspect K-15: Polypeptide according to any of aspects K-10 to K-14,    in which said one or more other binding units that provides the    polypeptide with increased half-life are chosen from the group    consisting of domain antibodies, immunoglobulin sequences that are    suitable for use as a domain antibody, single domain antibodies,    immunoglobulin sequences that are suitable for use as a single    domain antibody, “dAb”'s, immunoglobulin sequences that are suitable    for use as a dAb, or Nanobodies that can bind to serum albumin (such    as human serum albumin) or a serum immunoglobulin (such as IgG).-   Aspect K-16: Polypeptide according to aspect K-10 to K-15, in which    said one or more other binding units that provides the polypeptide    with increased half-life is a Nanobody that can bind to serum    albumin (such as human serum albumin) or a serum immunoglobulin    (such as IgG).-   Aspect K-17: Polypeptide according to any of aspects K-10 to K-16,    that has a serum half-life that is at least 1.5 times, preferably at    least 2 times, such as at least 5 times, for example at least 10    times or more than 20 times, greater than the half-life of the    corresponding immunoglobulin sequence according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1 per se or Nanobody according to any of aspects H-1 to    H-22 per se, respectively.-   Aspect K-18: Polypeptide according to any of aspects K-10 to K-17,    that has a serum half-life that is increased with more than 1 hours,    preferably more than 2 hours, more preferably more than 6 hours,    such as more than 12 hours, or even more than 24, 48 or 72 hours,    compared to the corresponding immunoglobulin sequence according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1    to E-13, F-1 to F-25 or G-1 per se or Nanobody according to any of    aspects H-1 to H-22 per se, respectively.-   Aspect K-19: Polypeptide according to any of aspects K-1 to K-18,    that has a serum half-life in human of at least about 12 hours,    preferably at least 24 hours, more preferably at least 48 hours,    even more preferably at least 72 hours or more; for example, of at    least 5 days (such as about 5 to 10 days), preferably at least 9    days (such as about 9 to 14 days), more preferably at least about 10    days (such as about 10 to 15 days), or at least about 11 days (such    as about 11 to 16 days), more preferably at least about 12 days    (such as about 12 to 18 days or more), or more than 14 days (such as    about 14 to 19 days).    Compound or Construct.-   Aspect L-1: Compound or construct, that comprises or essentially    consists of one or more immunoglobulin sequences according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1 and/or one or more Nanobodies according to    any of aspects H-1 to H-22, and optionally further comprises one or    more other groups, residues, moieties or binding units, optionally    linked via one or more linkers.-   Aspect L-2: Compound or construct according to aspects L-1, in which    said one or more other groups, residues, moieties or binding units    are immunoglobulin sequences.-   Aspect L-3: Compound or construct according to aspect L1 or L-2, in    which said one or more linkers, if present, are one or more    immunoglobulin sequences.-   Aspect L-4: Compound or construct according to any of aspects L1 to    L-3, in which said one or more other groups, residues, moieties or    binding units are immunoglobulin sequences.-   Aspect L-5: Compound or construct according to any of aspects L1 to    L4, in which said one or more other groups, residues, moieties or    binding units are chosen from the group consisting of domain    antibodies, immunoglobulin sequences that are suitable for use as a    domain antibody, single domain antibodies, immunoglobulin sequences    that are suitable for use as a single domain antibody, “dAb”'s,    immunoglobulin sequences that are suitable for use as a dAb, or    Nanobodies.-   Aspect L-6: Compound or construct according to any of aspects L1 to    L-5, in which said one or more immunoglobulin sequences of the    invention are immunoglobulin sequences.-   Aspect L-7: Compound or construct according to any of aspects L-1 to    L-6, in which said one or more immunoglobulin sequences of the    invention are chosen from the group consisting of domain antibodies,    immunoglobulin sequences that are suitable for use as a domain    antibody, single domain antibodies, immunoglobulin sequences that    are suitable for use as a single domain antibody, “dAb”'s,    immunoglobulin sequences that are suitable for use as a dAb, or    Nanobodies.-   Aspect L-8: Compound or construct, that comprises or essentially    consists of one or more Nanobodies according to any of aspects H-1    to H-22 and in which said one or more other groups, residues,    moieties or binding units are Nanobodies.-   Aspect L-9: Compound or construct according to any of aspects L-1 to    L-9, which is a multivalent construct.-   Aspect L-10: Compound or construct according to any of aspects L-1    to L-10, which is a multispecific construct.-   Aspect L-11: Compound or construct according to any of aspects L-1    to L-10, which has an increased half-life, compared to the    corresponding immunoglobulin sequence according to any of aspects    A-1 to A-22, B-1 to B-7. C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1 per se or Nanobody according to any of aspects H-1 to    H-22 per se, respectively.-   Aspect L-12: Compound or construct according to aspect L-1 to L-11,    in which said one or more other groups, residues, moieties or    binding units provide the compound or construct with increased    half-life, compared to the corresponding immunoglobulin sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se or Nanobody according    to any of aspects H-1 to H-22 per se, respectively.-   Aspect L-13: Compound or construct according to aspect L-12, in    which said one or more other groups, residues, moieties or binding    units that provide the compound or construct with increased    half-life is chosen from the group consisting of serum proteins or    fragments thereof, binding units that can bind to serum proteins, an    Fc portion, and small proteins or peptides that can bind to serum    proteins.-   Aspect L-14: Compound or construct according to aspect L-12 or L-13,    in which said one or more other groups, residues, moieties or    binding units that provide the compound or construct with increased    half-life is chosen from the group consisting of human serum albumin    or fragments thereof.-   Aspect L-15: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life are chosen from the group consisting of binding    units that can bind to serum albumin (such as human serum albumin)    or a serum immunoglobulin (such as IgG).-   Aspect L-16: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life are chosen from the group consisting of domain    antibodies, immunoglobulin sequences that are suitable for use as a    domain antibody, single domain antibodies, immunoglobulin sequences    that are suitable for use as a single domain antibody, “dAb”'s,    immunoglobulin sequences that are suitable for use as a dAb, or    Nanobodies that can bind to serum albumin (such as human serum    albumin) or a serum immunoglobulin (such as IgG).-   Aspect L-17: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life is a Nanobody that can bind to serum albumin    (such as human serum albumin) or a serum immunoglobulin (such as    IgG).-   Aspect L-18: Compound or construct according to any of aspects L-12    to L-17, that has a serum half-life that is at least 1.5 times,    preferably at least 2 times, such as at least 5 times, for example    at least 10 times or more than 20 times, greater than the half-life    of the corresponding immunoglobulin sequence according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to 0-6, E-1 to    E-13, F-1 to F-25 or G-1 per se or Nanobody according to any of    aspects H-1 to H-22 per se, respectively.-   Aspect L19: Compound or construct according to any of aspects L-12    to L-18 that has a serum half-life that is increased with more than    1 hours, preferably more than 2 hours, more preferably more than 6    hours, such as more than 12 hours, or even more than 24, 48 or 72    hours, compared to the corresponding immunoglobulin sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to 0-6, E-1 to E-13, F-1 to F-25 or G-1 per se or Nanobody according    to any of aspects H-1 to H-22 per se, respectively.-   Aspect L-20: Compound or construct according to any of aspects L-12    to L-19, that has a serum half-life in human of at least about 12    hours, preferably at least 24 hours, more preferably at least 48    hours, even more preferably at least 72 hours or more; for example,    of at least 5 days (such as about 5 to 10 days), preferably at least    9 days (such as about 9 to 14 days), more preferably at least about    10 days (such as about 10 to 15 days), or at least about 11 days    (such as about 11 to 16 days), more preferably at least about 12    days (such as about 12 to 18 days or more), or more than 14 days    (such as about 14 to 19 days).-   Aspect L-21: Monovalent construct, comprising or essentially    consisting of one immunoglobulin sequence according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to 0-6, E-1 to    E-13, F-1 to F-25 or G-1 and/or one Nanobody according to any of    aspects H-1 to H-22.-   Aspect L-22: Monovalent construct according to aspect L-21, in which    said immunoglobulin sequence of the invention is chosen from the    group consisting of domain antibodies, immunoglobulin sequences that    are suitable for use as a domain antibody, single domain antibodies,    immunoglobulin sequences that are suitable for use as a single    domain antibody, “dAb”'s, immunoglobulin sequences that are suitable    for use as a dAb, or Nanobodies.-   Aspect L-23: Monovalent construct, comprising or essentially    consisting of one Nanobody according to any of aspects H-1 to H-22.    Nucleic Acid-   Aspect M-1: Nucleic acid or nucleotide sequence, that encodes an    immunoglobulin sequence according to any of aspects A-1 to A-22, B-1    to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, a    Nanobody according to any of aspects H-1 to H-22.-   Aspect M-2: Nucleic acid or nucleotide sequence, that encodes a    compound or construct according to any of above aspects.    Host Cell-   Aspect N-1: Host or host cell that expresses, or that under suitable    circumstances is capable of expressing, an immunoglobulin sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1, a Nanobody according to any    of aspects H-1 to H-22, a polypeptide according to any of aspects    K-1 to K-19, a compound or construct according to any of aspects L-1    to L-21 that is such that it can be obtained by expression of a    nucleic acid or nucleotide sequence encoding the same, or a    monovalent construct according to any of aspects L-22 or L-23;    and/or that comprises a nucleic acid or nucleotide sequence    according to aspect M-1 or a genetic construct according to aspect    M-2.    Compositions-   Aspect O-1: Composition comprising at least one immunoglobulin    sequence according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to    C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, Nanobody according    to any of aspects H-1 to H-22, polypeptide according to any of    aspects K-1 to K-19, compound or construct according to any of    aspects L-1 to L-21, monovalent construct according to any of    aspects L-22 or L-23, or nucleic acid or nucleotide sequence    according to aspects M-1 or M-2.-   Aspect O-2: Composition according to aspect O-1, which is a    pharmaceutical composition.-   Aspect O-3: Composition according to aspect O-2, which is a    pharmaceutical composition, that further comprises at least one    pharmaceutically acceptable carrier, diluent or excipient and/or    adjuvant, and that optionally comprises one or more further    pharmaceutically active polypeptides and/or compounds.    Making of Agent and Composition of the Invention-   Aspect P-1: Method for producing an immunoglobulin sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1, a Nanobody according to any    of aspects H-1 to H-22, a polypeptide according to any of aspects    K-1 to K-19, a compound or construct according to any of aspects L-1    to L-21 that is such that it can be obtained by expression of a    nucleic acid or nucleotide sequence encoding the same, or a    monovalent construct according to any of aspects L-22 or L-23, said    method at least comprising the steps of:    -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence according to aspect M-1, or a genetic construct        according to aspect M-2;    -   optionally followed by:    -   b) isolating and/or purifying the immunoglobulin sequence        according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4,        0-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, a Nanobody        according to any of aspects H-1 to H-22, a polypeptide according        to any of aspects K-1 to K-19, a compound or construct according        to any of aspects L-1 to L-21, or a monovalent construct        according to any of aspects L-22 or L-23 thus obtained.-   Aspect P-2: Method for producing an immunoglobulin sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, 0-1    to 0-6, E-1 to E-13, F-1 to F-25 or G-1, a Nanobody according to any    of aspects H-1 to H-22, a polypeptide according to any of aspects    K-1 to K-19, a compound or construct according to any of aspects L-1    to L-21 that is such that it can be obtained by expression of a    nucleic acid or nucleotide sequence encoding the same, or a    monovalent construct according to any of aspects L-22 or L-23, said    method at least comprising the steps of:    -   a) cultivating and/or maintaining a host or host cell according        to aspect . . . under conditions that are such that said host or        host cell expresses and/or produces at least one immunoglobulin        sequence according to any of aspects A-1 to A-22, B-1 to B-7,        C-1 to C-4, D-1 to 0-6, E-1 to E-13, F-1 to F-25 or G-1,        Nanobody according to any of aspects H-1 to H-22, a polypeptide        according to any of aspects K-1 to K-19, a compound or construct        according to any of aspects L-1 to L-21, or monovalent construct        according to any of aspects L-22 or L-23;    -   optionally followed by:    -   b) isolating and/or purifying the immunoglobulin sequence        according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4,        0-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, Nanobody according        to any of aspects H-1 to H-22, a polypeptide according to any of        aspects K-1 to K-19, a compound or construct according to any of        aspects L-1 to L-21, or monovalent construct according to any of        aspects L-22 or L-23 thus obtained.        Method of Screening-   Aspect Q-1: Method for screening immunoglobulin sequences directed    against ion channels such as e.g. P2X7 that comprises at least the    steps of:    -   a) providing a set, collection or library of nucleic acid        sequences encoding immunoglobulin sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an        immunoglobulin sequence that can bind to and/or has affinity for        ion channels such as e.g. P2X7 and that is cross-blocked or is        cross blocking a Nanobody of the invention, e.g. SEQ ID NO: 705        to 788, more preferably SEQ ID NO's 726 to 750, 753 to 758, 762        to 764, 772 to 773, 775, or 778 to 780, more preferred SEQ ID        NO's 732, 773 or 778 (Table A-1) or a polypeptide or construct        of the invention, e.g. SEQ ID NO: 789 to 791 (see Table A-3);        and    -   c) isolating said nucleic acid sequence, followed by expressing        said immunoglobulin sequence.        Use of Binding Agent of the Invention-   Aspect R-1: Method for the prevention and/or treatment of at least    one [insert diseases and disorders], said method comprising    administering, to a subject in need thereof, a pharmaceutically    active amount of at least one immunoglobulin sequence according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E1    to E-13, F-1 to F-25 or G-1, Nanobody according to any of aspects    H-1 to H-22, polypeptide according to any of aspects K-1 to K-19,    compound or construct according to any of aspects L-1 to L-21,    monovalent construct according to any of aspects L-22 or L-23; or    composition according to aspect O-2 or 0-3.-   Aspect R-2: Method for the prevention and/or treatment of at least    one disease or disorder that is associated with ion channels such as    e.g. P2X7, with its biological or pharmacological activity, and/or    with the biological pathways or signalling in which ion channels    such as e.g. P2X7 is involved, said method comprising administering,    to a subject in need thereof, a pharmaceutically active amount of at    least one immunoglobulin sequence according to any of aspects A-1 to    A-22, B-1 to B-7. C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25    or G-1, Nanobody according to any of aspects H-1 to H-22,    polypeptide according to any of aspects K-1 to K-19, compound or    construct according to any of aspects L-1 to L-21, monovalent    construct according to any of aspects L-22 or L-23; or composition    according to aspect O-2 or 0-3.-   Aspect R-3: Method for the prevention and/or treatment of at least    one disease or disorder that can be prevented and/or treated by    administering, to a subject in need thereof, at least one    immunoglobulin sequence according to any of aspects A-1 to A-22, B-1    to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1,    Nanobody according to any of aspects H-1 to H-22, polypeptide    according to any of aspects K-1 to K-19, compound or construct    according to any of aspects L-1 to L-21, monovalent construct    according to any of aspects L-22 or L-23; or composition according    to aspect O-2 or O-3, said method comprising administering, to a    subject in need thereof, a pharmaceutically active amount of at    least one at least one immunoglobulin sequence according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1, Nanobody according to any of aspects H-1    to H-22, polypeptide according to any of aspects K-1 to K-19,    compound or construct according to any of aspects L-1 to L-21,    monovalent construct according to any of aspects L-22 or L-23; or    composition according to aspect O-2 or O-3.-   Aspect R-4: Method for immunotherapy, said method comprising    administering, to a subject in need thereof, a pharmaceutically    active amount of at least one immunoglobulin sequence according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1    to E-13, F-1 to F-25 or G-1, Nanobody according to any of aspects    H-1 to H-22, polypeptide according to any of aspects K-1 to K-19,    compound or construct according to any of aspects L-1 to L-21,    monovalent construct according to any of aspects L-22 or L-23; or    composition according to aspect O-2 or O-3.-   Aspect R-5: Use of an immunoglobulin sequence according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1, a Nanobody according to any of aspects H-1    to H-22, a polypeptide according to any of aspects K-1 to K-19, a    compound or construct according to any of aspects L-1 to L-21, or a    monovalent construct according to any of aspects L-22 or L-23 in the    preparation of a pharmaceutical composition for prevention and/or    treatment of at least one [insert diseases and disorders]; and/or    for use in one or more of the methods according to aspects R-1 to    R-3.-   Aspect R-6: Immunoglobulin sequence according to any of aspects A-1    to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1, Nanobody according to any of aspects H-1 to H-22,    polypeptide according to any of aspects K-1 to K-19, compound or    construct according to any of aspects L-1 to L-21, monovalent    construct according to any of aspects L-22 or L-23; or composition    according to aspect O-2 or O-3 for the prevention and/or treatment    of at least one disease or disorder in which an ion channel plays a    role or is implicated.    Fragment Aspects-   Aspect S-1: Part or fragment of an immunoglobulin sequence according    to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6,    E-1 to E-13, F-1 to F-25 or G-1, or of a Nanobody according to any    of aspects H-1 to H-22.-   Aspect S-2: Part or fragment according to aspect S-1, that can    specifically bind to ion channels such as e.g. P2X7.-   Aspect S-3: Part of fragment according to any of aspects S-1 or S-2,    that can specifically bind to ion channels such as e.g. P2X7 with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less,    and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably    10⁻⁸ to 10⁻¹² moles/liter.-   Aspect S-4: Part or fragment according to any of aspects S-1 to S-3,    that can specifically bind to ion channels such as e.g. P2X7 with a    rate of association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷    M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more    preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect S-5: Part or fragment according to any of aspects S-1 to S-4,    that can specifically bind to ion channels such as e.g. P2X7 with a    rate of dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹    preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between    10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect S-6: Compound or construct, that comprises or essentially    consists of one or more parts or fragments according to any of    aspects S-1 to S-4, and optionally further comprises one or more    other groups, residues, moieties or binding units, optionally linked    via one or more linkers.-   Aspect 3-7: Compound or construct according to aspect S-6, in which    said one or more other groups, residues, moieties or binding units    are immunoglobulin sequences.-   Aspect S-8: Compound or construct according to aspect S-6 or S-7, in    which said one or more linkers, if present, are one or more    immunoglobulin sequences.-   Aspect 3-9: Nucleic acid or nucleotide sequence, that encodes a part    or fragment according to any of aspects S-1 to S-7 or a compound or    construct according to aspect S-8.-   Aspect 3-10: Composition, comprising at least one part or fragment    according to any of aspects S-1 to 3-7, compound or construct    according to any of aspects 3-6 to S-8, or nucleic acid or    nucleotide sequence according to aspect S-9.    Derivatives Aspects-   Aspect T-1: Derivative of an immunoglobulin sequence according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to 0-6, E-1    to E-13, F-1 to F-25 or G-1, or of a Nanobody according to any of    aspects H-1 to H-22.-   Aspect T-2: Derivative according to aspect T-1, that can    specifically bind to ion channels such as e.g. P2X7.-   Aspect T-3: Derivative according to any of aspects T-1 or T-2, that    can specifically bind to ion channels such as e.g. P2X7 with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less,    and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably    10⁻⁶ to 10⁻¹² moles/liter.-   Aspect T-4: Derivative according to any of aspects T-1 to T-3, that    can specifically bind to ion channels such as e.g. P2X7 with a rate    of association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷    M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more    preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect T-5: Derivative according to any of aspects T-1 to T-4, that    can specifically bind to ion channels such as e.g. P2X7 with a rate    of dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably    between 10⁻² s⁻¹ and 10⁻⁶ s-more preferably between 10⁻³ s⁻¹ and 10⁶    s⁻¹, such as between and 10⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect T-6: Derivative of a polypeptide according to any of aspects    K-1 to K-19 or compound or construct according to any of aspects L-1    to L-23.-   Aspect T-7: Derivative according to aspect T-6, that can    specifically bind to ion channels such as e.g. P2X7.-   Aspect T-8: Derivative according to any of aspects T-6 or T-7, that    can specifically bind to ion channels such as e.g. P2X7 with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less,    and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably    10⁻⁸ to 10⁻¹² moles/liter.-   Aspect T-9: Derivative according to any of aspects T-6 to T-8, that    can specifically bind to ion channels such as e.g. P2X7 with a rate    of association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷    M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more    preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect T-10: Derivative according to any of aspects T-6 to T-9, that    can specifically bind to ion channels such as e.g. P2X7 with a rate    of dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably    between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and    10⁻⁶ s¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect T-11: Derivative according to any of aspects T-1 to T-10,    that has a serum half-life that is at least 1.5 times, preferably at    least 2 times, such as at least 5 times, for example at least 10    times or more than 20 times, greater than the half-life of the    corresponding immunoglobulin sequence according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1 per se, Nanobody according to any of aspects H-1 to H-22    per se, polypeptide according to any of aspects K-1 to K-19 or    compound or construct according to any of aspects L-1 to L-23 per    se.-   Aspect T-12: Derivative according to any of aspects T-1 to T-11,    that has a serum half-life that is increased with more than 1 hours,    preferably more than 2 hours, more preferably more than 6 hours,    such as more than 12 hours, or even more than 24, 48 or 72 hours,    compared to the corresponding immunoglobulin sequence according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1    to E-13, F-1 to F-25 or G-1 per se, Nanobody according to any of    aspects H-1 to H-23 per se, polypeptide according to any of aspects    K-1 to K-19 or compound or construct according to any of aspects L-1    to L-23 per se, respectively.-   Aspect T-13: Derivative according to any of aspects T-1 to T-12,    that has a serum half-life in human of at least about 12 hours,    preferably at least 24 hours, more preferably at least 48 hours,    even more preferably at least 72 hours or more; for example, at    least 5 days (such as about 5 to 10 days), preferably at least 9    days (such as about 9 to 14 days), more preferably at least about 10    days (such as about 10 to 15 days), or at least about 11 days (such    as about 11 to 16 days), more preferably at least about 12 days    (such as about 12 to 18 days or more), or more than 14 days (such as    about 14 to 19 days).-   Aspect T-14: Derivative according to any of aspects T-1 to T-13,    that is a pegylated derivative.-   Aspect T-15: Compound or construct, that comprises or essentially    consists of one or more derivatives according to any of aspects T-1    to T-14, and optionally further comprises one or more other groups,    residues, moieties or binding units, optionally linked via one or    more linkers.-   Aspect T-16: Compound or construct according to aspect T-15, in    which said one or more other groups, residues, moieties or binding    units are immunoglobulin sequences.-   Aspect T-17: Compound or construct according to aspect T-16, in    which said one or more linkers, if present, are one or more    immunoglobulin sequences.-   Aspect T-18: Nucleic acid encoding a compound or construct according    to aspect T-16 or T-17.-   Aspect T-19: Composition, comprising at least one derivative to any    of aspects T-1 to T-14, compound or construct according to any of    aspects T-15 to T-17, or nucleic acid or nucleotide sequence    according to aspect T-18.    Particularly Preferred Aspects-   1. Method for the generation of immunoglobulin sequences that can    bind to and/or have affinity for a cell-associated antigen    comprising the steps of:    -   a) genetic vaccination of a non-human animal with a nucleic acid        encoding said cell-associated antigen or a domain or specific        part of said cell associated antigen; and    -   b) optionally boosting the animal with said antigen in its        natural conformation selected from cells comprising natural or        transfected cells expressing the cell-associated antigen, cell        derived membrane extracts, vesicles or any other membrane        derivative harbouring enriched antigen, liposomes, or virus        particles expressing the cell associated antigen    -   c) screening a set, collection or library of immunoglobulin        sequences derived from said non-human animal for immunoglobulin        sequences that can bind to and/or have affinity for said        cell-associated antigen.-   2. The method according to aspect 1, wherein said cell-associated    antigen is selected from transmembrane antigens, including    transmembrane antigens with multiple spanning domains, including but    not limited to GPCRs or ion channels.-   3. The method according to aspect 1 or 2, wherein said non-human    animal is selected from vertebrates such as sharks, lizard, and    mammals, more specifically camelids such as llama and alpaca.-   4. The method according to any one of aspects 1 to 3, wherein the    non-human animal is a camelid or llama.-   5. The method according to any one of aspects 1 to 4, wherein said    immunoglobulin sequences are light chain variable domain sequences,    or heavy chain variable domain sequences.-   6. The method according to aspect 5, wherein the immunoglobulin    sequences are heavy chain variable domain sequences that are derived    from a conventional four-chain antibody or heavy chain variable    domain sequences that are derived from a heavy chain antibody.-   7 The method according to any one of aspects 1 to 6, wherein the    immunoglobulin sequences are domain antibodies, or immunoglobulin    sequences that are suitable for use as domain antibodies, single    domain antibodies, or immunoglobulin sequences that are suitable for    use as single domain antibodies, “dAbs”, or immunoglobulin sequences    that are suitable for use as dAbs, or Nanobodies, including but not    limited to V_(HH) sequences or immunoglobulin sequences that are    suitable for use as Nanobodies.-   8. The method according to aspect 7, wherein the immunoglobulin    sequences are Nanobodies.-   9. The method according to any one of aspects 1 to 8, wherein    vaccination is performed by a needle-free jet injection, by a    ballistic method, by needle-mediated injections such as Tattoo, by    topical application or by any DNA administration method followed by    in vivo electroporation.-   10. The method according to any one of aspects 1 to 9, wherein    vaccination is performed by intradermal, intramuscular or    subcutaneous administration of DNA.-   11. The method according to any one of aspects 1 to 10, wherein the    set, collection or library of immunoglobulin sequences is obtained    from the blood, lymph node, spleen, bone marrow or any tissue    harbouring cells encoding these immunoglobulin sequences of said    non-human mammal.-   12. The method according to any one of aspects 1 to 11, wherein said    cell-associated antigen is expressed on any cell which allows    expressing of the target in its native conformation such as but not    limiting to a cell selected from Cho, Cos7, Hek293, or camelid    derived cells such as Llama derived or Alpaca derived cell.-   13. The method according to any one of aspects 1 to 12, wherein said    cell-associated antigen is a membrane-spanning antigen such as e.g.    a GPCR and/or ion channel.-   14. The method according to any one of aspects 1 to 13, wherein said    antigen is selected from CXCR7, CXCR4 and/or P2X7.-   15. The method according to any of aspects 1 to 14, wherein the set,    collection or library of immunoglobulin sequences is expressed on a    set, collection or sample of cells or viruses and said set,    collection or sample of cells is screened for cells that express an    immunoglobulin sequence that can bind to and/or have affinity for    said cell-associated antigen.-   16. The method according to aspect 15, wherein a nucleic acid    sequence that encodes the immunoglobulin sequence that can bind to    and/or has affinity for said cell-associated antigen is purified    and/or isolated from the cell or virus, followed by expression of    said immunoglobulin sequence.-   17. The method according to any of aspects 1 to 16, wherein the set,    collection or library of immunoglobulin sequences is encoded by a    set, collection or library of nucleic acid sequences and said set,    collection or library of nucleic acid sequences is screened for    nucleic acid sequences that encode an immunoglobulin sequence that    can bind to and/or has affinity for said cell-associated antigen.-   18. The method according to aspect 17, wherein the nucleic acid    sequences that encode an immunoglobulin sequence that can bind to    and/or has affinity for said cell-associated antigen are purified    and/or isolated, followed by expressing said immunoglobulin    sequence.-   19. The method according to any one of aspects 1 to 18, wherein the    immunoglobulin sequence that can bind to and/or has affinity for    said cell-associated antigen is purified and/or is isolated.-   20. Immunoglobulin obtainable by a method of any one of aspects 1 to    19.-   21. Immunoglobulin directed against an ion channel obtainable by a    method of any one of aspects 1 to 19.-   22. Immunoglobulin according to aspect 21, wherein the    immunoglobulin is an antagonist (partial or full) of an ion channel.-   23. Immunoglobulin according to aspect 21, wherein the    immunoglobulin is an agonist (partial or full) of an ion channel.-   24. Immunoglobulin directed against P2X7 by a method of any one of    aspects 1 to 19.-   25. Composition comprising the immunoglobulin sequence according to    any of aspect 20 to 24.

EXAMPLES Example 1. Genetic Immunization of Llamas and Identification ofImmunoglobulin Sequences Using the Hepatitis B Small Surface Antigen asa Model Antigen

Target specific camelid immunoglobulin sequences were identifiedfollowing DNA vaccination. Hepatitis B small surface antigen was chosenas a model antigen, as this protein has been widely used to inducehumoral immune responses in animals following genetic immunization.

Example 1.1. Generation and Preparation of Plasmid for GeneticImmunization

Eukaryotic expression vector pRc/CMV-Hbs(s) encoding the Hepatitis Bsmall surface antigen (HBSAg) is obtained from Aldevron. Expression isunder control of the constitutive Cytomegalovirus (CMV) promoter. Thesequence of the resulting construct as been verified by sequenceanalysis.

Plasmid DNA is produced using Endotoxin-free Gigaprep kit (Qiagen)according to the manufacturer's instructions. The vector DNA is finallyreconstituted at a concentration of 1 mg/mL in endotoxin-free LAL H₂O orin endotoxin-free 0.9% NaCl in LAL H₂O. Plasmid is stored in aliquots at−20° C. Prior to use the plasmid DNA solution is centrifuged to removepossible aggregates.

Example 1.2. Induction of a Humoral Immune Response in Camelids ViaGenetic Immunization Following Distinct Methods of DNA Administration

After approval of the Ethical Committee of the Faculty of VeterinaryMedicine (University Ghent, Belgium), four llamas (124, 160, 117 and203) were immunized using two genetic immunization methods to induce anantigen specific humoral response. For both methods, DNA was appliedintradermally (ID). The first DNA administration method consisted ofneedle-free jet injection, the second followed a tattoo method (Bins etal. 2005. Nature Medicine 11:899-904).

Prior to the application of the DNA, an area of the llama skin ofapproximately 200-240 cm² is shaved above the shoulder-blade, the skinis treated with commercial depilation cream (Veet) for 2 minutes tochemically remove all remaining hair and part of the stratum corneum,and the shaved area is thoroughly cleaned by rinsing with water. For thefirst method, DNA is administrated into the skin using the Pig-jetdevice (Endoscopic)(llamas 124 and 160). A multi-nozzle head allows todistribute the DNA solution simultaneously over five adjacent spots of0.04 ml each, leaving injection blebs or papulae in the skin for acouple of hours. Each dose (1 mg DNA) the llama received (days 0, 14, 28and 57) thus resulted in 25 injection blebs. For the short-intervaltattoo method, a short-interval regimen was used. Llamas 117 and 203 areanaesthetized, and the area of DNA application is divided into threeparts for tattooing at days 0, 3, 7 (interval 1), 21, 24, 28 (interval2), 56, 59 and 63 (interval 3). One mg/ml droplets of DNA are appliedand tattooed into the skin using a commercial tattoo device (magnum 9formation needle) at 0.5 mm depth during at least 10 minutes per sessionover a surface of approximately 80 cm². The dose of administered DNA is1.33 mg (interval 1 and 2) and 4 mg (interval 3). From all llamas, smallblood samples are collected at regular intervals during the immunizationto monitor serum conversion via ELISA.

To verify whether the llamas induced a HBsAg specific humoral immuneresponse after DNA vaccination, an ELISA was executed with a 400-folddilution of pre-immune and immune sera. In brief, 1 μg/ml recombinantHBsAg (Aldevron) is immobilized overnight at 4° C. in a 96-well Maxisorpplate (Nunc). Wells are blocked with a casein solution (1% in PBS).After addition of the serum dilution, specifically bound immunoglobulinswere detected using a goat anti-llama-IgG horseradish peroxidaseconjugate (Bethyl Lab. Inc., Montgomery, Tex.). Results depicted inFIGS. 1A-1C demonstrate that for the jet injection method for bothllamas (124 and 160) a clear target specific serum conversion isdetected (day 0 vs day 80) although with variable magnitudes (FIGS. 1Aand 1B). After the third cycle of tattooing, a similar trend isdemonstrated for llama 117 and 203, although the magnitude of theresponse is lower compared to jet injection (FIGS. 1A and 1C).

Example 1.3. Boosting the DNA Primed Camelids with HBsAg ProteinIncreased Antigen Specific Serum Conversion, Including Heavy ChainAntibody Mediated Responses

At day 85, a single boost with 50 μg purified HBsAg using Stimune (CEDIDiagnostics, Lelystad, The Netherlands) as adjuvant was administeredintramuscularly (IM) into the neck of all four llamas, and small serumsamples were collected. Evaluation of the immune response for all fouranimals was performed via ELISA as described in the previous example andshowed that a single HBsAg boost induced a strong secondary response forall four animals (FIG. 2). Following this “DNA” prime-“protein” boostapproach, similar antigen specific serum titers were generated ascompared to an immunization method where only HBsAg protein has beeninjected (llamas 32 and 33; six weekly IM neck injections; dose of100-50 μg protein/injection using Stimune as adjuvant). Results areshown in FIG. 3. The antibody response was mounted both by theconventional and the heavy chain antibody expressing B-cell repertoires,since bound llama immunoglobulins were detected with monoclonalantibodies specifically recognizing the llama IgG1 conventionalantibodies or the heavy-chain only llama IgG2 and IgG3 antibodies (FIG.4)(Daley et al., Clin Diagn Lab Immunol. 2005 March; 12(3):380-6).

Example 1.4. Priming the Immune Response in Camelids Against HBsAg withDNA is Sufficient to Identify In Vivo Matured Antigen SpecificNanobodies

B-cell containing 150 mL blood samples were collected from llama 124 and117 (the llamas showing highest serum conversion following Pig-jet andTattoo DNA application, respectively) between the last DNAadministration and the HBsAg protein boost. Subsequently, peripheralblood lymphocytes (PBLs) were purified by a density gradientcentrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden)according to the manufacturer's instructions.

Total RNA was extracted and cDNA was prepared to amplify the Nanobodyrepertoire via nested PCR as previously described (e.g. WO 02/085945 andWO 04/049794). The PCR products were digested with SfiI (introduced vianested PCR in the FR1 primer region) and BstEII (restriction sitenaturally occurring in FR4) and following gel electrophoresis, the DNAfragment of approximately 350 bps was purified from gel. 330 ng ofamplified Nanobody repertoire was ligated into the correspondingrestriction sites of one μg of SfiI-BstEII digested phage display vector(pAX50) to obtain a library after electroporation of Escherichia coliTG1. pAX50 contains the LacZ promoter, a coliphage pill protein codingsequence, a resistance gene for ampicillin or carbenicillin, amulticloning site (harboring the SfiI and BstEII restriction sites) anda chimeric leader sequence consisting of gene3 and Erwinia carotovorapelB motifs. This display vector allows the production of phageparticles, expressing the individual Nanobodies as a fusion protein witha c-Myc, a His6-tag and with the geneIII product. The size of thelibraries derived from llama 124 and 117 immune tissues was 1×10⁸ and3×10⁷ CFUs respectively. As a library quality control, the percentage ofinsert of correct size was determined as 91 and 100%, respectively, by acolony PCR using the M13 reverse and a geneIII primer on 24 randomlypicked colonies of each library.

Libraries were rescued by growing the bacteria to logarithmic phase(OD₅₀₀=0.5), followed by infection with helper phage to obtainrecombinant phage expressing the repertoire of cloned Nanobodies on tipof the phage as a pill fusion protein. Phage was stored after filtersterilization at 4° C. for further use.

HBsAg specific Nanobodies were selected after a single round of panningas follows. Recombinant HBsAg (Aldevron) was directly immobilized onMaxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 500 and 50 ng perwell. After 2-hour incubation with the phage libraries and extensivewashing, bound phage was eluted with trypsin (1 mg/ml) during 15 minutesat room temperature. Protease activity was inhibited by adding 5 μl of16 mM ABSF protease inhibitor to the 100 μl of phage eluate. In allselection conditions, a higher number of eluted phage from a HBsAgimmobilized well was observed when comparing to the number of elutedphage eluted from a non-HBsAg coated well, indicating an enrichment forHBsAg specific Nanobodies. The output from each selection wasre-infected in logarithmically grown E. coli TG1 for 30 minutes at 37°C. and appropriate dilutions were grown overnight on solid medium (LBcontaining 2% glucose and ampicillin) to obtain single colonies.Individual colonies were picked from HBsAg enriched selection outputsand grown in 96 deep-well plates (1 ml volume) and induced by addingIPTG for Nanobody expression. Periplasmic extracts (PEs) were preparedin a volume of 80 μl according to standard methods.

In total 10-fold dilutions of 188 PEs (94 for llama 124 and 94 for llama117 derived PE) were screened for specific binding to solid-phase coatedHBsAg via ELISA, using mouse anti-Myc monoclonal antibody and subsequentstep anti-mouse-HRP conjugated detection antibodies. Periplasmicextracts showing minimal 2 fold signal above background (non-coatedwell) were scored as positive and corresponding Nanobody clones weresequenced. For libraries 124 and 117, 68% and 4% of the clones scoredpositive, respectively. Following sequence analysis, 5 HBsAg specificNanobody families were identified (3 from llama 124 and 2 from llama117; SEQ IDs Table 1.1) with representative family examples HBSAGPMP2E7(family 1), HBSAGPMP2E12 (family 2) and HBSAGPMP2A4 (family 3) fromllama 124 and HBSAGPMP1C6 (family 4) and HBSAGPMP1E11 (family 5) fromllama 117.

Example 1.5. Nanobodies Isolated from DNA Vaccinated Llamas Show SimilarOff-Rates to Those Identified from Protein Immunized Llamas

Plasma-derived HBsAg particles (Biodesign) were immobilized on surfaceplasmon resonance CM5 sensor chips (BIAcore) at a density of 11000 RUs.Regeneration of the chip surface was performed with a five second flowof 0.1 M HCl at a flow rate of 45 μl/minute. Periplasmic Nanobodyextracts were injected to evaluate the off-rates (Biacore). Data weredouble referenced by subtraction of the curves on the reference channeland of a blank running buffer injection. Processed curves were evaluatedby fitting a 1:1 dissociation model onto the binding curves in theBiacore T100 Evaluation software v1.1.1 and Biaevaluation software v4.1.Off-rates of HBSAGPMP2E7, HBSAGPMP2E12 were calculated as 8.8E-4 s−1 and1.3E-3 s⁻¹, respectively. These off-rates were similar to those obtainedfor the HBsAg specific Nanobodies identified after selection onlibraries obtained from llama 32 and 33 (off-rates between 6.0E-2 and1.7E-3 s⁻¹) (Serruys et al. 2009 Hepatology 49(1):39-49), indicatingthat the affinities of the Nanobodies obtained via genetic immunizationdo not differ from Nanobodies identified via protein immunization.

TABLE 1.1 Sequences: SEQ IDN Name NO: Immunoglobulin sequencePHbsAgPMP2E7 700 EVQLVESGGGLLQAGGSLRLSCAASERAFIIYGKAWFRQAPGKEREFVAGINWNGGDLHYADSVK GRFTISRDNTNNVVYLQMNSLKSEDTAVYYCAVRRGTAYETDVSSYEWGTQVTVSS PHbsAgPMP2E12 701EVQLVESGGGLVQAGGSLRLSCAASGRSISEYA MGWFRQAPGQEREFVASISTSGGSTTYADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCAR YNGWMYYAGTMGVHFGQGTQVTVSS PHbsAgPMP2A4702 EVQLVESGGGLVQPGGSLRLSCAASGSIDSINR MGWYRQAPGKQRELVASSTSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNF RGSYYSGYGDYWGKGTLVTVSS PHbsAgPMP1C6703 KVQLVESGGGWVRTGGSMRLSCAASGRTSSG SAMGWFRQAPGKERVFVAAISWGGAYTDYADSVKGRFTISRDNWRNTVDLQMNNLKPEDTAVYY CADGGSTWYEPTESDFGSWGQGTQVTVSSPHbSAgPMP1E11 704 EVQLVESGGGLVQPGGSLRLSCAASGSRDRLNVMGWYRQAPGKERDLVATMTAGGSTNYADSV KGRFTISRDIANMVYLQMNSLKPEDTAVYYCNGITASWYSGSYNYNNYWGQGTQVTVSS

Example 2. Identification of Nanobodies Against G-Protein CoupledReceptors Via Genetic Immunization

In order to demonstrate the feasibility of genetic immunization formembrane bound targets carrying multiple transmembrane domains, thehuman chemokine receptor CXCR4 was chosen as first example.

Example 2.1. Generation of a CXCR4-Encoding Vector Suitable for GeneticImmunization

Human chemokine receptor CXCR4 encoding cDNA (NM_003467) was purchasedfrom Open Biosystems. After PCR-mediated introduction of restrictionsites NheI (5′ end) and XhoI (3′ end), the amplicon was cloned into thecorresponding sites of pVAX-1 (Invitrogen) and pCDNA3.1 (Invitrogen).The sequence integrity of the resulting pVAX1-CXCR4 and pCDNA3.1-CXCR4was verified by sequence analysis. The vector pVAX1 was designed to beused for genetic immunization, and harbors the human cytomegalovirus(CMV) promoter. pVAX1 allows high-copy number replication in E. coli,transient high-level expression of the protein of interest in mostmammalian cells both in vitro and in vivo. Milligram amounts ofendotoxin-free pVAX1-CXCR4 plasmid was produced, dissolved to aconcentration of 2 mg/mL in 0.9% saline in LAL H2O and stored at −20° C.

Example 2.2. pVAX1-CXCR4 Transfected Cells Express Functional CXCR4Receptor

To verify the functional expression of human CXCR4, the endotoxin-freepVAX1-CXCR4 plasmid was transiently transfected into HEK293 cells usingEugene (Roche) to monitor in vitro extracellular expression via flowcytometry. Human CXCR4 specific monoclonal antibody 12G5 (R&D SystemsMAB170) followed by PE-labeled goat anti-mouse IgG detecting antibody(Jackson ImmunoResearch Inc. Cat. Nr. 115-115-164) were used as thedetection antibodies. Following this method, a 38-fold fluorescenceintensity shift of the transfected pVAX1-hCXCR4/HEK293 overnon-transfected HEK293 cells was shown (FIG. 5A). The presence offunctional CXCR4 at the cell surface was confirmed by binding of itsbiotinylated ligand CXCL12/SDF-1a (R&D Systems, Human CXCL12/SDF-1 alphaBiotinylated Fluorokine Kit NNS00) to CXCR4-transfected HEK293 but notparental HEK293 cells, following the manufacturers procedure (notshown).

Example 2.3. Generation of Stably Transfected CXCR4 Camelid Cells

For the generation of stable CXCR4 cell lines, pCDNA3.1-hCXCR4 plasmidwas transfected into immortalized camel kidney (CAKI) cells (Nguyen etal. 2001. Adv. Immunol. 79: 261-296). Individual transfected cellsshowing high expression density indicated by fluorescent staining withthe 12G5 antibody (as described in example 2.2) were cloned bydeposition of single cells in microtiter plate wells (FACSAria I withACDU, Becton Dickinson). After outgrowth of clonal cell lines in mediumcontaining selection antibiotics and confirmation of CXCR4 expressionvia flow cytometry (as in example 2.2), multiple homogenous stable CXCR4CAKI transfectants were obtained. One clone showing a fluorescence shiftof 114-fold over the non-transfected camelid cells, indicating highlevels of CXCR4 membrane expression, was selected for furtherexperiments (FIG. 5B).

Example 2.4. Intradermal Delivery of pVAX1-CXCR4 is Sufficient to Inducea Detectable Target Specific Humoral Immune Response in Llama

After approval by the ethical committee of the Faculty of VeterinaryMedicine, University Ghent, Belgium, four llamas (llama 389, 401, 402and 403) were assigned for genetic immunization. Immediately prior tothe administration of the CXCR4 encoding DNA, a skin area of 250 cm²covering the llama shoulder-blade was shaved and all remaining hairtissue was removed by application of commercial depilation cream asdescribed in Example 1.2. DNA dissolved into 0.9% saline wasadministered into the bald skin by needle-free jet injection using theautomatic dermojet named Vacci-jet (Akra DermoJet France) using a 3nozzle head. For all four DNA administrations (day 0, 14, 28 and 42),two mg DNA per llama was applied, distributed over multiple adjacentspots. Successful intradermal (ID) application of the DNA containingliquid is indicated by the formation of superficial liquid containingblebs or papulae on the skin for a couple of hours. Of each llama, apre-immune 10-ml serum sample and distinct serum samples during thegenetic vaccination procedure were collected to monitor the antigenspecific humoral response. Binding of llama immunoglobulins present inthe 250- to 5000-fold diluted pre-immune samples (day 0) and immuneserum sample (day 53; collected 11 days after the fourth DNAapplication) were scored for differential staining of CXCR4 transfectedcamelid versus non-transfected camelid cells via flow cytometry.Detection of cell bound llama total IgG (conventional+heavy-chainantibody) was detected via goat anti-llama IgG (Cat nr A160-100; Bethyl)followed by secondary staining with PE-conjugated donkey anti-goat IgG(Jackson ImmunoResearch Laboratories Cat nr. 115-115-164). Except forllama 403, a clear increase in mean cell fluorescence (MCF) was observedfor all day 53 immune sera on CXCR4-transfected CAKI cells as comparedto the pre-immune level (for minimally three dilutions tested), whileMCF values of non-transfected CAKI cells stained similarly remained low(FIGS. 6A and 6B). This indicated that following ID geneticimmunizations, 3 out of 4 llamas showed a specific humoral responseagainst the native target conformation.

Example 2.5. Genetic Immunization Followed by a Single Cell BoostSignificantly Increases CXCR4 Specific Serum Conversion

Camelid cells were chosen as the immunogen cell background to direct theimmune response towards CXCR4, anticipating reduced immunogenicity ofcamelid cell surface markers in llama due to high overall sequencesimilarity, as compared to human or rodent host cell backgrounds.Cultured CXCR4 expressing camelid cells (example 2.3) were freshlyharvested and washed twice with D-PBS to remove all culture mediumcontaminants. Cells were resuspended in 2 mL D-PBS and stored on iceduring transfer to the animal facilities. Llamas 389, 401, 402 and 403were subcutaneously (SC) injected with 2E7 hCXCR4 transfected camelidcells, minimally three weeks after the final DNA administration. Elevendays after the cell boost, an immune blood sample was collected fromeach llama and CXCR4 serum titer was determined as described in example2.4. For all four llamas (at 250 to 2250 fold serum dilutions),increased MCF values were detected on CXCR4 transfected camelid cellscompared to serum sample obtained after DNA administration alone (day53), while the serum binding to untransfected camelid cells was onlyslightly increased in three animals. These results indicate that thesingle cell boost resulted in an increased CXCR4 response magnitude forall four llamas (FIGS. 6A and 6B and Table 2.1). In parallel to the fourgenetic immunized llamas, the serum titer was determined on day 62samples collected from llamas 217 and 218 (FIGS. 6A and 6B and Table2.1). These llamas were immunized with six CXCR4-HEK293 cell injections(1-4E7 cells per dose) at weekly intervals (described in patentWO/2009/138519A1). Only one of the two llamas (218) showed a strongserum response to CXCR4-expressing cells compared to the untransfectedcontrol cells (n=2; one representative example shown). The detected MCFvalues indicated that the executed genetic immunization procedure(DNA+cell boost) generates a similar or better target specific titermagnitude compared to a full cell immunization

TABLE 2.1 Discovery overview of CXCR4 specific Nanobody B-cell lineages.Number of CXCR4 specific Nanobody CXCR4 Specificity families (some ofLlama response Selection screening which are displacing ID Immunogen(FACS) Library conditions Hit-rate (ELISA) ligand) 389 DNA + DNA R1: 10U18% (8/45)  5 R2: 10U 9% (4/45) R2: 1U 13% (6/45)  hCXCR4/camelid ++ PBR1: 10U 4% (2/45) 3 cells R2: 10U 76% (34/45) R2: 1U 78% (35/45) 401DNA + DNA R1: 10U 0% (0/45) 3 R2: 10U 2% (1/45) R2: 1U 4% (2/45)hCXCR4/camelid ++ PB R1: 10U 22% (10/45) 3 cells R2: 10U 89% (40/45) R2:1U 76% (34/45) 402 DNA + DNA R1: 10U 0% (0/45) 4 R2: 10U 16% (7/45)  R2:1U 7% (3/45) hCXCR4/camelid ++ PB R1: 10U 36% (16/45) 22 cells R2: 10U67% (30/45) R2: 1U 67% (30/45) 403 DNA − DNA R1: 10U 4% (2/45) 6 R2: 10U31% (14/45) R2: 1U 27% (12/45) hCXCR4/camelid + PB R1: 10U 7% (3/45) 4cells R2: 10U 51% (23/45) R2: 1U 44% (20/45) 217 hCXCR4/Hek293 + CellsR1: 10U 4% (2/45) 10 cells R2: 10U 89% (40/45) R2: 1U 78% (35/45) 218hCXCR4/Hek293 + Cells R1: 10U 7% (3/45) 15 cells R2: 10U 93% (42/45) R2:1U 96% (43/45)

Example 2.6. Genetic Immunization is Sufficient to Identify CXCR4Specific Nanobodies

From llamas 389, 401, 402, 403, four 150 ml blood samples were collected3 and 9 days following the last DNA administration (encoded PBL1 andPBL2, respectively) and 4 and 8 days after the cell boost (PBL3 andPBL4, respectively). Additionally, a biopsy of the palpable bow lymphnode (LN) was collected from each llama via local surgery three to fourdays after the cell boost. Peripheral blood lymphocytes were purifiedfrom PBL1-4 samples by density gradient centrifugation on Ficoll-Paqueas described in Example 1.4. From all lymphocyte harboring immunetissues total RNA was extracted and used as template to prepare cDNA (asdescribed in example 1.4). For each genetic immunized llama, 2 separatelibraries were generated (Table 2.2): one library derived from pooledPBL1+2 cDNA (‘DNA’ library) and a second one derived from pooled PBL3+4and LN (post boost or ‘PB’ library).

TABLE 2.2 Genetic distance of five in vivo matured CXCR4 Nanobodyfamilies versus parental V-germline sequences. Nanobody Average targetAverage target family interaction interaction Average number Averagenumber originated Library (number potency potency of nt mutations of AAmutations Nanobody from of Nanobody (MCF ratio; (absorption ratio;versus parental versus parental Family Llama variants) FACS) ELISA)V-germline¹ V-germline¹ A 389 DNA (1) 46 14 19 10 PB (16) 553 33 26 12 B389 DNA (1) 105 7 17 12 PB (3) 261 11 19 13 C 389 DNA (2) 505 43.5 5.5 5PB (6) 556 87.3 13 9 D 402 DNA (1) 755 56 21 15 PB (7) 671 73 22 14.6 E403 DNA (2) 381 20.5 17 10.5 PB (2) 412 10.5 18 11.5 ¹Any change,including a deletion or an addition are considered to calculate thenumber of mutations.

Though the polyclonal serum responses detected via flow cytometryindicate the presence of target specific llama antibodies against nativeCXCR4, the magnitude of the titer is not necessarily predictive for i)the anti-CXCR4 heavy-chain antibody mediated clonal diversity, ii) theaffinity of monoclonal Nanobodies for the target and iii) of theresponse width, e.g. the CXCR4 epitopes covered by the individualNanobodies. CXCR4-specific Nanobodies were identified via phage displayin order to characterize these on a monoclonal level. Parallelselections were performed on each of the eight DNA and PB libraries, andon two additional libraries generated from cell immunized llamas.Libraries were generated similarly in a similar manner from pooled cDNAderived from PBL1+2, collected 4 and 8 days after the final cellinjection.

To select CXCR4 specific Nanobodies, recombinant phage was rescued fromall ten libraries as described under example 1.4. In a first selectionround, 10 units of 96-well Maxisorp plate (Nunc) immobilized membranevesicles derived from CXCR4 transfected HEK293 cells were blocked withlow-fat milk powder (Marvell 4% in PBS). After 2 hours of incubationwith rescued phage, trypsin elution (1 mg/ml) was allowed for 15 minutesat room temperature subsequent to 15 PBS washes. Protease activity wasimmediately neutralized by applying 0.8 mM protease inhibitor ABSF. Allphage outputs were infected into logarithmically grown E. coli TG1 cellsand were plated on agar plates (LB+Amp+2% glucose) for analysis ofindividual Nanobody clones. The round 1 phage outputs were rescued and asecond selection round on 10 or 1 units of plate-immobilized CXCR4membrane vesicles was performed. Enrichment was calculated as the ratiobetween the number of phage eluted from CXCR4 membrane vesicles versusthose eluted from non-transfected HEK293 control membrane vesicles. For8 out of 10 libraries, round two outputs showed enrichments >5 (data notshown). The round 2 phage outputs selected on 10 or 1 units plateimmobilized hCXCR4 membrane vesicles were infected into TG1 cells andwere plated on agar plates (LB+Amp+2% glucose). Forty five individualclones of each output selected on CXCR4 Membrane vesicles (round 1 andround 2) were grown in 1-ml 96-deep-well plates and periplasmic extracts(PEs) were prepared as described under example 1.4.

CXCR4 specificity was determined via two screening assays using twodifferent receptor formats. In a first method, two units of CXCR4Membrane vesicles were immobilized per well on 96-well Maxisorp platesby overnight coating at 4° C. Following blocking with 4% Marvel in PBS,10-fold diluted PE was added and bound Nanobody (harbouring a c-Myc tag)was detected via sequential mouse anti-Myc and rabbit anti-mouse-HRPdetection. 501 Nanobodies, showing a minimally 5-fold increased ELISAsignal on hCXCR4 membrane vesicles over control membrane vesicles, wereconsidered to be CXCR4 specific. The corresponding average ratios of thetwo types of negative control PEs, one generated from TG-1 expressing anirrelevant Nanobody and another from TG-1 containing an empty expressionvector, were 1.1±0.8 and 1.2±0.7, respectively. Resulting from thisscreening method, hit-rates for each selection output were calculatedand are summarized in Table 2.1. Based on the average hit-rates on round2 selection outputs (10 U membrane vesicles) following the differentimmunization strategies, discovery efficiencies were calculated as 14,71 and 91% for ‘DNA’, ‘PB’ and ‘cell’ repertoires, respectively (FIG.7). Even in absence of a detectable heavy chain antibody (HcAb) titer toCXCR4 (Table 2.1), target specific Nanobodies were identified, asindicated by hit-rates of 4 to 31% after one and two rounds of selectionon llama 403 DNA library, respectively. The hit-rates indicate thatafter the single cell boost, a single round of selection was sufficientto identify CXCR4 specific Nanobodies from all HcAb llama repertoires(hit-rates in Table 2.1 between 4 and 36%).

All 501 CXCR4 specific Nanobodies were sequenced and redundantNanobodies (identical AA sequence) were removed. This resulted in theidentification of 171 unique sequences, belonging to 70 distinctNanobody B-cell lineages (Table 2.1). Nanobodies belong to the sameB-cell lineage or family when their CDR3 region show high amino acidsequence similarity and are of the same length. The CDR3 is anticipatedto contribute mostly to the antigen interaction and consequentlyNanobodies belonging to the same family are assumed to bind the sameepitope. The average number of CXCR4 specific Nanobody familiesidentified per llama is 12.5 after cell immunizations (217, 218) and11.2 via DNA immunization (DNA+cell boost; 389, 401, 402 and 403),respectively. The number of Nanobody amino acid sequence variants(minimally 1 AA residue mutation) belonging to one Nanobody family wasin the range of 1 to maximally 17 family variants.

CXCR4 specific binding was confirmed in a secondary screening assaymeasuring Nanobody binding to cells expressing human CXCR4 via flowcytometry. Hereto, five-fold diluted PEs were incubated with parental orCXCR4 transfected camelid cells (2×10⁵ cells) and Nanobody binding wasmeasured via mouse anti-Myc (Serotec MCA2200) and subsequent anti-mouseIgG-PE (Jackson ImmunoResearch Laboratories 115-115-164) detectionantibodies. For all samples, the ratio of the MCF value on CXCR-4expressing cells to the MCF on untransfected control cells wascalculated. While an irrelevant Nanobody consistently showed a ratio<2.4, a ratio >10 was detected for 61 Nanobody families (at least onefamily member), thereby confirming specificity of these families fornative CXCR4. Of the remaining nine Nanobody families, seven familiesconsisting of a single member (7 families) showed a ratio <3, despiteabsorbance ratios of 5-10 fold measured in ELISA on Membrane vesicles.For the remaining two Nanobody families (2 families; all single memberfamilies) the absorbance ratios (ELISA) were 144 and 70, respectively,while the MCF ratios (FACS) were 2.2 and 2.3, respectively.

Example 2.7. Intradermal DNA Administration as Immunization Method isSufficient to Identify Nanobodies Modulating SDF-1-CXCR4 ReceptorInteraction

Subsequent to the screening for CXCR4 specificity (determined via ELISAor FACS), all 171 CXCR4 specific Nanobody variants were tested for theirability to block the interaction of the ligand SDF-1 with its receptorto identify receptor function modulating Nanobodies (as described inpatent WO/2009/138519A1). In brief, 40 μM of [¹²⁵] SDF-1 ligand(in-house labelled) was allowed to bind 2 μg of hCXCR4/HEK293 membraneextracts in presence or absence of Nanobody competitor ten-fold dilutionof a PE (produced as described in example 1.4). After incubation for 1hour at 4° C., membrane extracts were washed and the total amount ofbound ligand radioactive counts per minute (cpm) was determined.Aspecific binding of the radio-labelled ligand to the membrane extracts(non-CXCR4 related) was determined by addition of excess unlabelledSDF-1 (100 nM) to compete all radio-ligand from the CXCR4 receptor. Theaspecific binding value for each plate was subtracted from the totalbinding (cpm in absence of NB) and the cpm values obtained for eachNanobody, and % residual [¹²⁵I] SDF-1 binding (SDF_(res)) in presence ofNanobody was calculated. A number of Nanobody families scored as liganddisplacer were identified (Table 2.1), showing that ligand competingNanobodies were identified from each immunization strategy (representingrepertoires DNA, PB and cell).

Example 2.8. Epitope Mapping of Nanobodies Identified after theDifferent Immunization Strategies

A selection of target specific Nanobodies (competitors and binders) fromeach immunization strategy will be recloned in an expression vectorallowing expression and purification of soluble Nanobody fused to a His6and Myc tag for further characterization. Expression will occur in E.coli after IPTG induction at 37° C. After spinning the cell cultures,periplasmic extracts will be prepared by freeze-thawing the pellets andresuspension in dPBS. These extracts are used as starting material forimmobilized metal affinity chromatography (IMAC). Nanobodies will beeluted from the column using 250 mM imidazole and subsequently desaltedtowards dPBS. Purity and integrity of all Nanobodies will be verified bypolyacrylamide gel electrophoresis (SDS-PAGE), while the presence oftags will be verified by western blotting.

To determine whether the DNA vaccination strategy (DNA only or followedby cell boost) results in the identification of Nanobodies againstdifferent epitopes compared to the Nanobodies identified after the cellimmunization strategy, a number of epitope binning assays will beimplemented. These are: binding to the CXCR4 N-terminal peptidecorresponding to AA residues 1-14 (ELISA), binding to CXCR4 mutantreceptors in which point-mutations within the N-terminus and theextracellular loop regions have been introduced, or binding to mutatedCXCR4 such as N-terminally truncated hCXCR4 receptor or CXCR4 chimerasin which the N-terminus or extracellular loops have been individuallyreplaced by the sequence of a related GPCR. Alternatively, epitopemapping will be performed using competition experiments of our panel ofpurified Nanobodies with distinct CXCR4-specific compounds, including i)monoclonal antibodies or Fab fragments of which the epitope has beendescribed (Carnec et al. 2005, J. Vir. 1930-33) such as 4G10 (SantaCruz, SC53534) which binds to the linear N-terminus, ii) small moleculeinhibitors, such as the antagonist AMD3100 (Sigma A5602), and iii) otherNanobodies labeled with a fluorescent marker or biotin.

Example 2.9. Cell Boost Following Genetic Immunization Generates aDifferent Nanobody Repertoire

From llamas 389, 401, 402 and 403, 45 CXCR4 specific Nanobody familieswere identified consisting of 123 Nanobody sequence variants (out of 225sequenced). Each of these 123 variants was assigned to the ‘DNA’libraries or the ‘PB’ library (no variant with identical AA sequence wasobtained from both libraries). Only for five of these 45 families (11.1%of the total family diversity), Nanobody variants belonging to the samefamily were identified from the ‘DNA’ and ‘PB’ libraries derived fromthe same animal (Table 2.2). These five families comprise 42 differentNanobody variants, corresponding to 34% of the non-redundant clones. Theremaining Nanobody repertoire (76%) is identified either from the ‘DNA’library or from the ‘PB’ library indicating that the cell boostfollowing to genetic immunization caused in vivo maturation of aNanobody repertoire not readily identified via panning of a librarygenerated after genetic immunization only (FIG. 8). Genetic immunizationonly results in a different Nanobody repertoire, indicating thatrepertoire may be lost after the cell boost.

Example 2.10. The Average Potency of the ‘DNA’ Repertoire is Lower thanthat of the ‘PB’ and ‘Cell’ Repertoire

The previous example indicates that only a limited number of familieshas been identified from both ‘DNA’ and ‘PB’ libraries. In order toverify what the effect is of the cell boost for this repertoire subset,we scored in vivo maturation by i) calculating the genetic distanceversus the parental V-gene germline sequence (in number of amino acid ornucleotide mutations; excluding the D- and J-gene segments encoding theCDR3 and FR4 region) for each variant within the specific Nanobodyfamily and ii) by comparing Nanobody variant potencies. For thecalculation of the genetic distance, we assumed a proportionalintroduction of amplification errors for all libraries (a proofreadingpolymerase has been used to limit the number of mutations caused by theNanobody repertoire cloning method including amplification via PCR). Forthree of these five families, the ‘B’ originating Nanobodies showed onaverage 12, 13 and 9 AA mutations (or 26, 19 and 13 nt mutations) versusthe parental V-gene germline sequence, while respectively 10, 12 and 5(or 19, 17 and 5.5 on the nt level) for the Nanobodies derived from the‘DNA’ library, which suggests that on average the ‘B’ originatingNanobodies are more distant from the parental V-gene germline (hencemore matured) than the ‘DNA’ originating Nanobodies. For these threefamilies, the higher degree of maturation of the ‘PB’ Nanobodies is alsoreflected in the average binding potency (MCF ratio as described inexample 2.6) of the ‘PB’ versus ‘DNA’ originating Nanobodies beingrespectively 553 vs 46, 261 vs 105, 556 vs 505 for 3 familiesrespectively (FACS results but trend is confirmed in ELISA, see Table2.2).

When analyzing concentration dependent target binding for all 122 uniqueNanobodies from llama 389, 401, 402 and 403 (20 and 102 Nanobodyvariants originating respectively from the ‘DNA’ and ‘PB’ libraries),the average MCF ratios (FACS) were 427 and 196 for the Nanobodiesoriginating from ‘PB’ and ‘DNA’ libraries, respectively (FIGS. 9A and9B). The corresponding number calculated from the 49 Nanobody variantsoriginating from ‘cell’ libraries (llamas 217 or 218) is 538. This trendis confirmed via the numbers generated by the ELISA assay: the averageadsorption ratios (calculated as described under example 2.6) were 36.2and 23 for the Nanobodies originating from ‘PB’ and ‘DNA’ libraries,respectively. The corresponding number calculated from the 49 Nanobodyvariants originating from ‘cell’ libraries (llamas 217 or 218) is 28.This suggests that the average genetic distance of the Nanobody variantsoriginating from the ‘DNA’ libraries are more closely related to theparental germline sequence than those Nanobodies identified from ‘PB’libraries, hence considered as being less matured. Moreover, whenanalyzing the average binding potencies of the three repertoires, the‘DNA’ repertoire appears less potent than the ‘PB’ repertoire, thelatter being similarly potent to the ‘cell’ repertoire (FIGS. 9A and9B).

For the fourth family however, surprisingly, the variants obtained fromthe ‘DNA’ library suggested an equal to higher degree of in vivomaturation compared to the Nanobodies identified from the ‘PB’ libraries(Table 2.2). The ‘DNA’ originating clone from this family shows 15 AA(or 21 nt) mutations compared to the V-germline and the correspondingaverage value for the ‘PB’ originating Nanobodies is 14.6 (AA) or 21.7(nt). This is also reflected in the average potencies in the FACS assayof the ‘DNA’ and ‘PB’ Nanobodies, being 755 and 671, respectively. Forthe fifth family, maturation of the ‘DNA’ and ‘PB’ libraries is scoredas highly similar since no consistent ranking between the ‘DNA’ and ‘PB’originating Nanobodies is detected following the 4 analysis methods (AAor nt mutations, FACS and ELISA).

Although there is no statistically significant difference betweenaverage binding potencies of the three different repertoires, theaverage potency of the ‘PB’ repertoire is similar to the one obtainedfrom the ‘cell’ libraries, which are higher than this of the ‘DNA’repertoire. On the monoclonal level however, Nanobodies with similarbinding potencies have been identified after genetic immunization onlycompared to those identified after cell boost or a cell immunization.

Example 3. Identification of Nanobodies Against a Ligand-Operated IonChannel

As a second example to demonstrate the feasibility of geneticimmunization for membrane bound targets carrying multiple transmembranedomains, the mouse purinoceptor P2X7 was chosen.

P2X7 is a ligand-gated ion channel, which is activated by highconcentrations of exogeneous ATP or by NAD-dependent ADP-ribosylation.The functional channel is formed by three P2X7 protein subunits, eachconsisting of two membrane-spanning regions and a single extracellularloop of 285 AA residues. Activation of the purinoceptor induces aconformational change, leading to the formation of a large non-selectivepore, ultimately causing membrane blebbing and apoptosis.

Genetic immunization with P2X7 has previously been demonstrated tosuccessfully raise polyclonal and monoclonal anti-P2X7 antibodies inrabbits and rats (Adriouch et al, 2005. Cell Immun 236, 72-77). Here wedemonstrate the identification of P2X7-specific Nanobodies that modulatethe ligand-induced P2X7 activation following genetic immunization.

Example 3.1. Induction of a Humoral Response in Llama to mP2X7 ViaGenetic Immunization Using a Ballistic Method

Gene gun immunization has been shown to be an efficient method ofintradermal DNA delivery for the induction of a humoral response in avarious range of animals, including mouse, bovine and llama (Koch-Nolte2007, Faseb. J. 21, 3490-3499). This ballistic administration methoddelivers DNA coated gold microparticles to the highly immune competentskin tissue under high pressure, immediately targeting and transfectingimmune effector cells present in the dermis such as the Langerhansantigen presenting cells. This results in increased in vivo transfectionefficiency and consequently a sustained antigen presentation consideredto stimulate the induction of humoral immune responses.

Three llamas (407, 414 and 417) were immunized with mouse P2X7 (mP2X7)encoding DNA. The device used for the DNA delivery was the HeliosGene-gun (Biorad). Endotoxin-free mP2X7-expression plasmidpCDNA6.1-mP2X7 (Adriouch et al. 2005, Cell Immun 236, 72-77) was coatedonto 1 μm gold particles (Biorad, cat nr. 1652263) following themanufacturer's instructions. Llama skin in the neck region was preparedas in example 1.2. Each llama received four antigen doses withadministration intervals of two weeks. Each dose consisted of 12 shotsof plasmid-conjugated gold particles (1 μg of DNA conjugated onto 0.5 mggold particles per shot) applied with a pressure setting at 600 psi intothe skin. Three weeks after the final genetic immunization, all llamasreceived a single boost with 2×10⁷ mP2X7-transfected Hek293 cells. Atregular intervals, blood samples were collected to monitor the inductionof the humoral immune response over time. For the isolation of B-celltissues, blood was collected from these animals 3 and 9 days after thefourth DNA immunization (PBL1 and PBL2), and 4 and 8 days after the cellboost (PBL3 and PBL4). A biopsy of the palpable lymph node (LN) in thebow area was taken 4 days after the cell boost.

Another three llamas (413, 415 and 416) were immunized subcutaneously inthe bow area with 2×10⁷ stably transfected mP2X7 Hek293 cells (Adriouchet al. 2005, Cell Immun 236, 72-77) for four times with two weekintervals. Blood was collected from these animals 4 days and 8 daysafter the fourth immunization and a LN biopsy was taken 4 days after thefourth immunization.

The serum response was monitored for mP2X7 reactivity via flow cytometryusing untransfected and mP2X7-transfected CHO cells similarly asdescribed in example 2.4. For the DNA immunized llamas, the mP2X7 serumconversion was compared between the pre-immune serum sample (day 0), aserum sample collected after the final DNA immunization (day 51, PBL2)and one after the cell boost (day 71, PBL4). For the llamas immunizedvia four cell injections, mP2X7 specific titers were compared betweenthe pre-immune and an immune sample collected at day 52. FIG. 10 showsthe total IgG (conventional and heavy-chain antibody) immune response ofall six mP2X7 immunized llamas. Except for llama 407, a clear increasein mean cell fluorescence (MCF) of mP2X7-transfected CHO cells wasobserved with the day 71 immune serum sample collected after the cellboost, compared to the pre-immune level (for minimally three dilutionstested). MCF values against non-transfected CHO cells remain low.Following the genetic immunization procedure (DNA priming followed bysingle cell boost), two out of three llamas showed a specific humoralresponse against the native target conformation. All three animalshaving received multiple cell injections show detectable mP2X7 specificserum titers (day 52). The background response against irrelevant CHOcell surface antigens is higher in these animals than in those immunizedvia genetic immunization (DNA+cell boost).

Example 3.2. Genetic Cocktail Immunization of Human and Mouse P2X7 UsingGene Gun

One benefit of genetic immunization is the versatility to generateimmune response against multiple targets simply by administeringdifferent target encoding genes to the same animal. Cocktailimmunizations can also be used to bias the immune response to a specificconformation. As example of cocktail immunizations, we chose human (h)and mP2X7, orthologues which share 80.5% overall sequence identity asillustrated in FIG. 11, for Gene gun DNA immunization of llamas. Inaddition, to allow induction of an immune response to the P2X7 channelin its open conformation, two modifications to the immunization strategydescribed in example 3.1 were made. At first, a cocktail immunizationwas applied with a mixture of plasmids encoding for mP2X7 and mArt2.2,which is a known activator of P2X7 by mediating ADP-ribosylation.Second, prior to the cell boost the P2X7-transfected Hek293 cells weretreated with ATP to activate the channel, after which cells were fixedusing paraformaldehyde to preserve the open conformation.

Two llamas (405 and 418) were immunized simultaneously with both h andmP2X7 using genetic immunization with the Gene gun. A mixture ofendotoxin-free pCDNA6.1-mP2X7 and pCDNA6.1-mArt2.2 (1:10 ratio) wasconjugated to 1 μm gold particles. Similarly, the pCDNA6.1-hP2X7 plasmidencoding hP2X7 was conjugated to 1 μm gold particles. Geneticimmunization was performed as described under example 3.1 (1 μg/0.5mg/shot). The left flank of the neck was used for immunization withmP2X7/mArt2.2-conjugates, while the right flank was used forimmunization with hP2X7 conjugates. Three weeks after the fourth DNAimmunization, both llamas were boosted simultaneously with 2×10⁷ATP-treated, fixed mP2X7-Hek293 cells (left side) and hP2X7-Hek293 cells(right side). A second boost was given at the left side after 3 dayswith 2×10⁷ ATP-treated, fixed hP2X7-Hek293 cells. For the isolation ofB-cell tissues, blood was collected from these animals 3 and 9 daysafter the fourth DNA immunization (PBL1 and PBL2), and 4 and 8 daysafter the first cell boost (PBL3 and PBL4). A LN biopsy was taken fromthe bow area at the left side 3 days after the first cell boost.

The total IgG serum titer response to mP2X7 of both llamas was monitoredin flow cytometry using untransfected and mP2X7-transfected cells, asdescribed in example 3.1. Hereto the pre-immune serum sample (day 0) wascompared to samples taken after DNA immunization (day 51, PBL2) and therespective cell boost (day 71, PBL4). Llama 418 shows a clear total IgGimmune response to mP2X7 after DNA immunization, which is furtherenhanced after the cell boost, while llama 405 only shows mP2X7reactivity after the cell boost (FIG. 12). For both llamas, the MCFvalues on untransfected CHO cells remain low. The serum reactivity tohuman P2X7 was not determined. In conclusion, after genetic immunizationand subsequent cell boost 4 out of 5 llamas show clear mP2X7 serumresponses.

Example 3.3. Induction of a Heavy-Chain Antibody Response after GeneticImmunization

In addition, the induction of a heavy-chain antibody (HcAb) mediatedmP2X7-specific response was monitored by FACS using llama IgGisotype-specific monoclonal antibodies on stable Hek293-mP2X7transfectants. To test this setup, dilutions of llama sera (1/200 forDNA-immunized llamas and 1/1000 for cell-immunized llamas) werepre-adsorbed on 1×10⁷ WT Hek293 cells at room temperature for 1 hour todeplete for cell background antibodies. Next, 5×10⁵ Hek293-mP2X7 oruntransfected Hek293 cells were incubated with 0.1 mL of thepre-adsorbed sera for 0.5 hour at 4° C. (to prevent internalization),after which bound HcAbs were detected by a mixture of mouse-anti-llamaIgG2 and IgG3 antibodies (Daley et el. 2005. Clin Diagn Lab Immunol12:380-386) followed by anti-mouse IgG-PE (Jackson ImmunoResearch)staining. Binding of conventional llama antibodies was detected usinganti-llama IgG1. As control for mP2X7 staining, the rat mAb Hano43 wasincluded (Adriouch et al. 2005, Cell Immun 236, 72-77). mP2X7 specificIgG serum titers of Hek293-mP2X7 immunized llamas could not bedetermined due to high levels of Hek293 cell background binding, whileno such high background was observed for DNA-immunized llama 407. FIGS.13A and 13B show the conventional antibody (FIG. 13A) and HcAb mediated(FIG. 13B) serum response after genetic immunization with mP2X7 (llama414 and 417) or the cocktail of m and hP2X7 (llama 405 and 418). AfterDNA vaccination only, sera of llamas 414 and 418 showed a clear MCFshift on mP2X7 transfected Hek293 cells vs non-transfected WT Hek293cells for the conventional Ab-mediated response, confirming the serumconversions detected via total IgG (in example 3.1 and 3.2). The cellboost induced a moderate (llama 417) to clear (llamas 405, 414 and 418)conventional Ab mediated MCF shift on mP2X7 transfected Hek293 cells vsnon-transfected WT Hek293 cells. A moderate HcAb response was detectedfor llamas 414 and 418 before the cell boost, which was enhanced forllama 414 by the cell boost (compare day 71 and day 67 sera). Incontrast, there was only little if any detectable heavy chain serumresponse for llamas 405 and 417 (FIG. 13B). In conclusion, in two out offive llamas, genetic immunization resulted in a detectable HcAb responseto mP2X7 after cell boost.

Example 3.4. Library Construction

Peripheral blood mononuclear cells were prepared from blood samplesusing Ficoll-Hypaque according to the manufacturer's (AmershamBioscience) instructions. Next, total RNA was extracted from these cellsas well as from the LN biopsy and used as starting material for RT-PCRto amplify Nanobody encoding gene fragments (example 1.4). For the fiveDNA immunized llamas 405, 407, 414, 417 and 418, cDNA obtained from PBL1and PBL2 after DNA immunizations only were used to generate ‘DNA’libraries, while the cDNA from PBL3, PBL4 and LN was used to constructpost-boost (PB) libraries. For the llamas immunized with Hek293-mP2X7cells, cDNA obtained from PBL1, PBL2 and LN was used for the generationof libraries. Fragments corresponding to the variable domain of the HcAbrepertoire were cloned into phagemid vector derived from pUC119 whichcontains the LacZ promoter, a E. coli phage pIII protein codingsequence, a resistance gene for ampicillin or carbenicillin, amulticloning site and the gene3 leader sequence. In frame with theNanobody coding sequence, the vector codes for a C-terminal c-myc tagand a (His)₆ tag. In total 13 phage libraries were generated, designated405-DNA; 405-PB, 407-DNA, 407-PB, 414-DNA, 414-PB, 417-DNA, 417-PB,418-DNA 418-PB, 413-cell, 415-cell, and 416-cell. The size of theresulting phage libraries was estimated between 0.12-2.5×10⁸ clones withinsert percentages between 83-100% Phage was prepared according tostandard methods and stored at 4° C. for further use.

Example 35. Nanobody Selection on mP2X7-Expressing Cells Using PhageDisplay

To identify Nanobodies recognizing the mP2X7 purinoceptor in its nativeconformation, selections were performed on whole cells expressing mP2X7using all 10 libraries generated from animals after DNA vaccination andthree libraries generated from the cell-immunized llamas. In order tocompare the characteristics of the monoclonal Nanobodies identified viadifferent immunization methods, selections on all libraries wereperformed in parallel. In a first round of selection, CHO cellstransfected with mP2X7 or untransfected CHO cells (5×10⁶ per library)were blocked with blocking buffer (10% fetal bovine serum (FBS) and 2%Marvel in PBS) for 30 minutes at 4° C. As preblocking step, all phageinputs were incubated in blocking buffer for 30 minutes at 4° C. Cellswere incubated with the phage under slow rotation at 4° C., then spundown and washed three times with blocking buffer and twice more usingPBS. Cell-bound phage were eluted using trypsin, as in example 1.4. Allphage outputs were infected into logarithmically growing E. coli TG1cells and plated on agar plates (LB+Amp+2% glucose) for analysis ofindividual Nanobody clones. Enrichment was calculated as the ratiobetween the number of phage eluted from mP2X7-CHO cells versus thoseeluted from non-transfected CHO cells in parallel selections. Firstselection round enrichments higher than 3-fold could only be observedfor 413 and 414 libraries, the PB library of the latter even showing anenrichment >200-fold. Round 1 mP2X7/CHO selected phage outputs wererescued and used for a second selection round on mouse Yac-1 cells,which endogenously express mP2X7. The change in cellular background wasdone to reduce the selection of phage binding to irrelevant cellbackground markers. Essentially the same procedure was followed as forround 1. Since no control cells were available for this second round,relative enrichments were calculated by taking the ratio of the round 2to round 1 outputs. From the DNA libraries, only for 407 and 417enrichments were detected (ratio of 6 and 97, respectively). For the PBrepertoire, 3 out of 5 libraries showed enrichments above 10, while for407 a moderate 3-fold enrichment was observed. No further enrichment wasseen for PB library 414, which may be due to the already high antigenspecific phage enrichment during the first selection round. From thecell-immunized libraries, only 413 showed a moderate 3-fold enrichment.Forty five to 48 individual clones of each selection output (round 1 andround 2) were grown in 96-well formats and phage were prepared forsubsequent screening, as described under example 1.

Example 3.6. Screening for mP2X7-Specific Nanobodies

P2X7 specificity was at first determined by phage ELISA on untransfectedand mP2X7-transfected CHO cells. Cells were seeded in 96-well cultureplates at density of 4×10⁴ cells per well in 0.2 ml of DMEM mediumcontaining 10% FBS supplemented with penicillin and streptomycin (100U/ml and 100 μg/ml, respectively) and incubated for 24 hours at 37° C.in a 5% CO₂ atmosphere. Sub-confluent cells were rinsed with PBS andfixed using 4% paraformaldehyde in PBS for 10 minutes at RT. Fixed cellswere washed three times with PBS, and blocked with 2% BSA in PBS for 2hours. Following blocking, 10-fold diluted culture supernatantcontaining phage was added and incubated for 1 hour. Cell-bound phagewas detected via mouse anti-M13-HRP-conjugate (GE Healthcare, Cat nr.E2886). Rat mAb Hano43 was used as positive control. 523 out of 1506screened individual clones derived from all selection rounds showed aminimally 2-fold increased ELISA signal on mP2X7-CHO cells relative tocontrol CHO cells. Hit-rates after primary screening of DNA, PB, andcell libraries are shown in table 3.1.

Based on the average hit-rates on round 1 and 2 selection outputs onrepertoires generated after the different immunization strategies (DNA,PB, cell), discovery efficiencies were calculated as 4, 40 and 11 (round1 outputs) or 31, 78, and 60% (round 2 outputs) for ‘DNA’, ‘PB’ and‘cell’ repertoires, respectively (FIG. 14). This indicated that theproportion of mP2X7 binding Nanobodies in the different repertoires washigher for ‘PB’ and ‘cell’ than for ‘DNA’. For cDNA immunized animals, agood correlation between serum titer and hit-rate after two selectionrounds was observed for PB libraries from llamas 405, 418 and 414, withhit-rates ranging from >48% after round 1 and >90% after round 2. Forcomparison, the hit-rates observed with libraries from cell immunizationranged between 40-73% after two selection rounds. Despite the absence ofa detectable serum titer in Hamas 407 and 417 after DNA immunization(FIG. 10), the hit-rate of the corresponding DNA libraries was 25% and87%, respectively, after 2 selection rounds. As shown previously forCXCR4, this indicates DNA vaccination is sufficient to identifymP2X7-specific Nanobodies via the phage display technology, even in theabsence of a detectable target-specific serum conversion.

Example 3.7. Sequence Diversity of mP2X7-Binding Clones

The sequences of all mP2X7 binders in the primary screening wasanalysed. Out of 523 Nanobody clones, 84 unique clones were identifiedthat could be categorised into 29 different sequence families (FIG. 22).The number of Nanobody amino acid sequence variants belonging to thesame family is summarized in FIG. 15. Respectively 15 and 14 familieshave been identified after DNA vaccination (DNA+PB) and cellimmunization, resulting in an average diversity of 3 families afterDNA+cell boost immunization method and 4.7 after the cell immunizationstrategy. Four mP2X7 families were identified that comprised familyvariants from both DNA and PB libraries, i.e. families 2, 15, 25 and 5(FIG. 14). Two of these clones, 16D9 (family 15, llama 414) and 6A11(family 5, llama 418), were identified from both the DNA and PBrepertoire. However, as shown earlier for CXCR4, most families wereidentified exclusively in the ‘DNA’ (4 out of 15 families) or the ‘PB’libraries (7 out of 15 families). This indicates that the cell boostfollowing genetic immunizations caused in vivo maturation of a Nanobodyrepertoire not readily identified in a library generated after geneticimmunizations only (Table 3.1).

TABLE 3.1 Screening hit rates after mP2X7 genetic and cell immunization.Serum titer Primary screening NB Llama Immunization FACS hit-rate (%)families ID strategy Library (total IgG) R1 R2 identified 407 DNA (4x)DNA − 0.0 11.1 25  mP2X7/HEK PB − 2.2 39.1 6, 25, 28 (1x) 414 DNA (4x)DNA +/− 6.7 11.1 15, 26, 27, 29 mP2X7/HEK PB ++ 73.9 93.5 1, 4, 15 (1x)417 DNA (4x) DNA − 11.1 86.7 2 mP2X7/HEK PB +/− 2.2 60.9 2, 24 (1x) 405Cocktail DNA DNA − 0.0 0.0 — (4x) m + hP2X7/HEK PB + 47.9 97.9 7, 16(1x) 418 Cocktail DNA DNA + 2.1 50.0 5, 17 (4x) m + hP2X7/HEK PB ++ 70.8100.0 5 (1x) 413 mP2X7/HEK Cell + 27.1 72.9 3 (4x) 415 mP2X7/HEK Cell +2.1 68.8 8, 10, 11, (4x) 12, 14, 18, 19, 20, 21 416 mP2X7/HEK Cell + 4.239.6 9, 13, 22, (4x) 23

Example 3.8. Confirmation of Specificity and Ranking of mP2X7-BindingClones for Selection of Representative Clones for Each Family

All non-redundant mP2X7-specific clones were re-arrayed in 96-wellplates for the production of PEs in 1 mL 96-deep-well plates, asdescribed in example 1. To confirm that the mP2X7 specific Nanobodiesidentified after screening for binding to fixed cells can also bindnative mP2X7, PE of all non-redundant clones were analysed for directbinding to mP2X7-Hek293 cells in FACS using the myc-tag for detection.Ten-fold diluted PE were pre-incubation with 0.5 μg anti-mycFITC-conjugated antibodies (AbD Serotec, cat no. MCA2200F) (creatingpseudo-bivalent Nanobodies in order to increase binding strength) in 0.1ml PBS/10% FCS for 45 minutes at 4° C. PE dilutions were incubated witha mixture of Hek293-mP2X7 and untransfected cells (5×10⁵ of each) for0.5 hour at 4° C., after which the cells were rinsed and cell-boundfluorescence was detected by means of flow cytometry. Alternatively,sequential staining of 10-fold diluted PE with anti-myc FITC-conjugatedantibodies was done after incubation with the cells. For members ofNanobody family 1, 2, 4, 5, 25 (from DNA/PB libraries) and 10 and 11(from cell immunization libraries), a clear avidity benefit was seen,indicating that the epitopes of these Nanobodies are still readilyaccessible for pseudo-dimerized Nanobodies. The effect of pre-incubationfor Nanobodies 2C4 (family 1) and 5A1 (family 3) is illustrated in FIG.16. Of each family, the best binder was selected for furthercharacterization. Families 6, 26-29 only showed low binding tomP2X7-expressing cells, even after pre-incubation with anti-Mycantibodies and were thus excluded from further characterization. Fromfamilies with unique clones derived from both DNA and PB libraries(families 2, 5, 15, and 25), at least two representatives were selectedto allow comparison of DNA and PB-derived clones. In turn, this wouldallow determining the possible contribution of the cell boost onpotencies and affinities of the respective Nanobodies. The panel ofselected Nanobodies for further characterization is depicted in FIG. 17and Table 3.2.

TABLE 3.2 Characterization of mP2X7-specific Nanobodies. Titration NBTitration ligand Fold Cho IC50 (nM) IC50 (nM) ATP IC50 NAD IC50 P2X7/wtagainst against (mM) by (mM) by P2X7 (phage 100 uM ATP 20 uM NAD 2 mM NB22 mM NB Clone Family Llama Library function ELISA) n = 2 n = 2 n = 1 n= 1 2C4 1 414 PB 4 2A6 1 414 PB 8 1A8 2 417 DNA 20 20C9 2 417 PB 11 7E23 413 Cell 25 4B4 4 414 PB enhancer 19 47 5 8G11 5 418 PB block 26 37 68389 >540 8F5 5 418 DNA block 16 350  579  171 partial 8G12 5 418 PBblock 36 55 82 290 >540 8C7 7 405 PB part.block 21 386* 4000*  8E7 7 405PB part.block 16 576* 4000*  7D6 8 415 Cell enhancer 18 7A4 9 415 Cell32 7B4 10 415 Cell 23 7H6 11 415 Cell block 23  122.6  208.3 494 >5407G5 12 415 Cell enhancer 30 13B8 13 416 Cell 24 7D5 14 415 Cell 14 1G615 414 DNA 12 16D9 15 414 DNA, PB 7 6B7 16 405 PB part.block 24 14D5 17418 DNA enhancer 8 16 2 13G5 18 415 Cell 35 13B5 19 415 Cell part.block28 436  3653  200 partial 13F6 20 415 Cell 20 13G4 21 415 Cell 56 13A722 416 Cell block 25  6 11 565 >540 13G9 23 416 Cell block 17 35 136 469 >540 20A11 24 417 PB 11 18C1 25 407 DNA 18 18A7 25 407 PB 14*Nanobodies were tested n = 1

Example 3.9. Production and Purification of mP2X7-Specific Nanobodies

The encoding sequences of the selected Nanobodies were recloned in anexpression vector derived from pUC119 which contained the LacZ promoter,a resistance gene for kanamycin, a multicloning site and the OmpA signalpeptide sequence (as described in example 2.8). In frame with theNanobody coding sequence, the vector coded for a C-terminal c-myc tagand a (His)6 tag. Expression occurred in E. coli TG-1 cells as c-myc,His₆-tagged proteins in a culture volume of 250 mL. Expression wasinduced by addition of 1 mM IPTG and allowed to continue for 3 hours at37° C. After spinning the cell cultures, periplasmic extracts wereprepared by freeze-thawing the pellets and resuspension in dPBS. Theseextracts were used as starting material for immobilized metal affinitychromatography (MAC) using Histrap FF crude columns (GE Healthcare,Uppsala, Sweden). Nanobodies® were eluted from the column with 250 mMimidazole and subsequently desalted towards dPBS. Purity and integrityof all Nanobodies was verified by polyacrylamide gel electrophoresis(SDS-PAGE), while the presence of tags was verified by western blottingusing anti-His antibodies for detection.

Example 3.10. None of the Mouse P2X7 Nanobodies are Cross-Reactive toHuman P2X7 or to P2X4

Concentration dependant mP2X7 binding of purified Nanobodies wasanalysed using stable mP2X7-expressing CHO cells in FACS. Serialdilutions of Nanobodies ranging from 1.2 uM to 6.7 μM were prepared andincubated with 2×10⁵ cells in 0.1 ml dPBS supplemented with 10% FBS(FACS buffer) for 0.5 hour at 4° C. Next, cells were washed three times,and bound Nanobody was detected using mouse anti-Myc (Serotec MCA2200)and subsequent staining with anti-mouse IgG-PE (Jackson ImmunoResearchLaboratories 115-115-164). As positive control, hybridoma supernatant ofthe rat Hano43 antibody was used, which was visualized by anti-ratIgG-PE (Jackson ImmunoResearch Laboratories 112-116-143). Results aredepicted in FIG. 17. The maximum MCF value obtained with an irrelevantcontrol Nanobody ranged between 314 and 355 (concentrations between 1.2μM to 6.7 μM). For Nanobodies 7D5 (family 14), 13A7 (family 22) and 7E2(family 3), EC₅₀ values are 3, 3.4 and 4.3 nM, respectively. For allother Nanobodies tested, no sigmoidal curve was obtained andconsequently no EC₅₀ values could be determined (FIG. 17). Of note, thestrongest binding Nanobodies were all derived from the three llamasimmunized with Hek293-P2X7 cells. For all Nanobodies specific bindingwas confirmed, except for 2C4, 20A11 and 13F6 for which no MCF valueabove 600 were detected.

Second, the specificity of Nanobodies of all 24 families was determinedby analysis of cross-reactive binding to Hek293 cells expressing thehuman orthologue of P2X7, and CHO cells expressing hP2X4, another memberof the P2X purinoceptor family (Möller et al Purinergic Signal3:359-366, 2007). Of each Nanobody 250 ng (final concentration of 147nM) was pre-incubated with anti-Myc-FITC conjugated antibodies,following the same procedure as described in example 3.7 except that nountransfected Hek cells were included. As positive controls, antibodiesL4 (specific for hP2X7, Buell et al. Blood 15: 3521-3528) and CR29(specific for hP2X4, Möller et al Purinergic Signal 3:359-366, 2007)were used, while an irrelevant Nanobody was included as negativecontrol. None of the Nanobodies was found to bind hP2X7-Hek293 orhP2X4-expressing CHO cells. Despite the fact that some Nanobodies wereisolated from llamas immunized with both h and mP2X7 (Families 5, 7, 16,and 17), none of these were cross-reactive to hP2X7.

Example 3.11. Identification of Nanobodies which Enhance or Block mP2X7Receptor Function

All purified Nanobodies from 24 different families were analysed fortheir ability to modulate the functional activity of the mP2X7purinoceptor. Activation of P2X7 occurs by extracellular ATP (directactivation) or NAD-dependent ADP-ribosylation (indirect activation) andresults in calcium release, exposure of phosphatidyl serine and sheddingof CD62L, ultimately leading to apoptosis (Scheuplein et al. 2009, J.Immun. 182(5):2898-908). Ligand-induced CD62L-shedding was used todetermine whether Nanobodies could interfere with mP2X7 function.

In a first approach, 5×10⁵ Yac-1 cells (endogenously expressing mP2X7)were incubated with a single concentration of Nanobodies (5 μg, finalconcentration of 2 μM), in RPMI supplemented with 10% FBS for 15 minutesat RT. Subsequently the P2X7 ion channel was activated by addition ofATP to a final concentration of 100 μM, or NAD to a final concentrationof 20 μM, followed by further incubation of the cell suspension for 20minutes at 37° C. Previous titration experiments indicated that theseligand concentrations induced maximal CD62L-ectodomain cleavage. Cellswere washed and the presence of cell-surface CD62L was detected in FACSusing anti-CD62L-PE conjugated antibodies (BD Pharmingen, cat no.553151). The percentage of CD62L-positively stained cells was taken asmeasure for modulation of P2X7 function. As controls, no Nanobody orirrelevant control Nanobody were included, which resulted inCD62L-shedding by 80-98% of cells, i.e. 2-20% of cells remaining CD62Lpositive, depending on the ligand and individual experiment. A Nanobodywas considered a mP2×7 blocker when the percentage of cells remainingCD62L positive was more than 3-fold the value obtained with theirrelevant Nanobody. Ten Nanobodies, representing seven families(families 5, 7, 11, 16, 19, 22, and 23), blocked both ATP and NADinduced CD62L shedding, with the two Nanobodies of family 7 lying justabove the threshold. Furthermore, two Nanobodies belonging to families16 and 19 appeared to block the NAD-induced but not the ATP-inducedresponse, suggesting these may selectively target the NAD-dependentADP-ribosylation site on mP2X7 (Adriouch et al. FASEB J 22:861-869,2008). In order to confirm inhibitory capacities, as well as to rank theNanobody blocking potencies in the ATP and NAD mediated CD62L sheddingassays, 9 out of these 10 Nanobodies were tested in serial dilutions intwo parallel experiments. A representative example is shown in FIG. 18.Inhibition of P2X7 function could be confirmed for all blockers in bothligand mediated assays and IC50 values are presented in Table 3.2. Thesuggested NAD-selectively for family 19 Nanobody 13B5 was not confirmed.Based on blocking potency, Nanobodies can be ranked as follows:13A7>8G11, 13G9, 8G12>7H6>13B5, 8F5>8C7, 8E7.

To verify if Nanobodies by themselves could activate the mP2X7 channel,cells were also incubated with 2 μM Nanobodies without subsequent ligandtreatment. None of the tested monovalent Nanobodies was able to directlyactivate mP2X7. However, for certain Nanobodies an increase in CD62Lectodomain shedding was observed after nucleotide treatment, suggestingthat these clones may facilitate the gating of mP2X7. Enhancement ofATP- and NAD-induced P2X7 activation was verified using sub-optimalligand concentrations: below 100 μM for ATP and below 20 μM for NAD.Yac-1 cells were treated with a fixed concentration of Nanobody (2 μM)and next stimulated with increasing concentrations of ATP (1-2700 μM) orNAD (1-540 μM). In case of ATP, three Nanobodies were found to clearlyenhance the CD62L shedding in the following order of efficacy:14D5>4B4=7D6 (FIGS. 19A-19D). For NAD the enhancement was less apparent.

Blocking Nanobodies were also tested in the same assays (at a fixedconcentration of Nanobody vs. increasing concentrations of ligand). Atthe highest concentration of NAD tested (540 μM), most blockers werestill maximally inhibiting cleavage, with the exception of 13B5 and 8F5which reached a plateau around 60% inhibition (FIGS. 19A-19D). Based onthe titration curve obtained with ATP the following ranking in blockerscould be made; 13A7, 13G9, 7H6>8G11, 8G12>8F5, 13B5 (Table 3.2).Comparing the three family 5 Nanobodies, both clones derived from the PBlibraries (8G11 and 8G12) were superior blockers to the clone from theDNA library (8F5).

Receptor modulating Nanobodies (blockers and enhancers) were identifiedfrom all three repertoires ‘DNA’, ‘PB’ or ‘Cell’ with the most potentblacker, 13A7, (IC50s of 6 nM and 11 nM in ATP and NAD assay,respectively) identified from a ‘Cell’ library. In contrast, the mostpotent enhancer, 14D5, was identified from a ‘DNA’ library.

Example 3.12. Multivalent Nanobodies with Improved Receptor ModulationPotencies

Bivalent Nanobody constructs, consisting of a head-to-tail geneticfusion of two identical Nanobody sequences connected by a 35 amino acidGlySer linker were generated from the functional blocking Nanobodies,13A7 (family 22, Cell) and 8G11 (family 5, PB), as well as from partialagonist 14D5 (family 17, DNA). Constructs were made by means of separatePCR reactions (one for the N-terminal, and one for the C-terminalNanobody subunit) using different sets of primers encompassing specificrestriction sites. An expression vector derived from pUC119 was usedwhich contained the LacZ promoter, a resistance gene for kanamycin andthe OmpA signal peptide sequence. Directly downstream of the signalpeptide a multiple cloning site was present for Nanobody insertion,followed by a 35Gly-Ser linker encoding DNA sequence and a secondmultiple cloning site for cloning of a second Nanobody sequence. Inframe with the resulting Nanobody-35Gly-Ser-Nanobody coding sequence,the vector coded for a C-terminal c-myc tag and a (His)6 tag. Afterverification of the nucleotide sequences, all three bivalent mP2X7Nanobody constructs were expressed and purified. Production was done inE. coli TG1 cells, followed by purification from the periplasmicfraction via the His-tag by IMAC and desalting, essentially as describedin example 3.9.

The potencies of monovalent and bivalent 14D5 in enhancing mP2X7activity were compared by measuring the enhancement of CD62L-ectodomainshedding at suboptimal nucleotide concentrations (33 μM ATP, 1.5 μMNAD), where no ectodomain shedding occurs in Yac-1 cells (as describedin example 3.11). The increase in potency of bivalent 14D5 vs.monovalent 14D5 was approximately 220-fold for shedding induced by ATP(EC₅₀ of 0.1 vs. 22.6 nM) and 40-fold for NAD (EC₅₀ 0.06 vs. 4.1 nM). Inthe absence of ligand, neither monovalent nor bivalent 14D5 could inducegating of P2X7.

As illustrated in FIGS. 20A-20D, the potencies of the two blockingNanobodies 13A7 and 8G11 were determined at 100 μM ATP and 20 μM NAD,respectively. Bivalents of 13A7 and 8G11 had potencies in thesub-nanomolar range for both ATP and NAD. The potency increase ofbivalent 13A7 vs. monovalent 13A7 was only moderate, with a 23 foldincrease in case of NAD and a 9-fold increase in case of ATP-inducedshedding (IC₅₀ bivalent 0.1 nM for ATP, and 0.3 nM for NAD). For 8G11the potency increase from mono- to bivalent was much stronger, with146-fold (NAD) and 84-fold (ATP)(IC₅₀ bivalent 0.2 nM for ATP, and 0.52nM for NAD).

Example. 3.13 Nanobodies Map to Different Epitopes on the mP2X7 Trimer

Recently, the crystal structure of zebrafish purinoceptor P2X4 waspublished (Kawate et al. 2009. Nature 460:592-598). Based on theavailable P2X4 structure (pdb3I5D and pdb3H9V with resolutions at 3.5and 3.1 Å, respectively), a model for mP2X7 was built using homologymodeling software (Modeler built in Discovery Studio, Accelrys). Thesequence identity between zebrafish P2X4 and mP2X7 comprises 49% in theextracellular region. First, the sequence of the mouse P2X7 was alignedwith the sequence of the known P2X4 structure. The sequence alignmenttakes care of a proper positioning of the gaps between the 2 sequencesand of the conserved disulfide bonds. Second, the 3D coordinates of theknown P2X4 structure are used to predict those of the unknown mP2X7. Aprobability density function (pdf) describes the restraints on thegeometric features and is used to evaluate the 3D model. Out of anexisting panel of mP2X7 arginine mutants that show extracellular mP2X7expression to a similar level as mP2X7 WT, seven mutants were selected(Adriouch et al. 2008, FASEB J 22:861-869, Schwarz et al. PurinergicSignal 5:151-161, 2009). According to the generated model, these sevenmutants represent six structurally dispersed regions on the mP2X7molecule; mutant R125A (site I; R125 residue critical for NAD mediatedribosylation), R294A (site I; cleft located residue critical for ATPbinding), R206A (site II; gain-of-function mutant located near theinterface of two mP2X7 interacting monomers); R151A (site III; criticalresidue for binding anti-mP2X7 mAb Hano44; Adriouch et al. 2008, FASEB J22:861-869); R178A (site IV); R230A (site V); R53K (site VI; residuelocated near the transmembrane domain at the interface of two mP2X7interacting monomers). In order to determine whether binding of theNanobodies to mP2X7 might be affected by these substitutions, 24purified Nanobodies representing different families were evaluated forbinding to non-transfected Hek293 cells and Hek293 cells transientlytransfected with WT or mutant mP2X7 receptors. For flow cytometricassays, detection of Nanobodies was performed via anti-Myc andsubsequent goat anti-mouse-PE conjugate, while an estimation of P2X7expression densities was obtained via staining with rat monoclonalHano43 followed by secondary anti-rat-PE conjugate. After normalizationof expression densities over the different transient transfectants (WTmP2X7 and 7 mutants) by subtraction of background binding to Hek293cells, residual binding of the different Nanobodies at 1 μM werecalculated.

A reduction of binding >90% was only detected for six Nanobodies, all tothe mutant R151A: Nanobody 7H6 (cell, 415, fam 11), 7D5 (cell, 415, fam14), 6B7 (PB, 405, fam 16), 7E2 (cell, 413, fam 3), 8011 (PB, 418, fam5), 8012 (PB, 418, fam 5). This implicates that all other Nanobodies arenot critically dependent on Arg151 for binding, either because they bindto separate regions, or that these Nanobodies do not bind to the Argresidue within their footprint. As expected, mP2X7 specific rat antibodyHano44 did not show any binding to R151A mutant.

Example 3.14. The Average Potency of the ‘DNA’ Repertoire is Comparableto that of the ‘PB’ but Lower than that of the ‘Cell’ Repertoire

Only four families were identified from both ‘DNA’ and ‘PB’ libraries.We scored in vivo maturation for each variant within the specificNanobody family relative to the closest related Llama V-germlinesequence (comprising of FR1 to FR3) as in example 2.10 and comparedNanobody variant potencies (Table 3.3). For family 2, 5, 15 and 25, the‘PB’ originating Nanobodies showed on average 19.5, 16.5, 10 and 14 AAmutations versus the parental V-gene germline sequence, whilerespectively 19.5, 17, 10.3 and 15 for the Nanobodies derived from the‘DNA’ library, which argues for equal maturation of the ‘DNA’ and ‘PB’originating Nanobodies. When the sequences of the CDR3 regions of DNAand PB derived family members are compared, an increase in diversity inthe PB relative to the DNA sequences is observed only for families 5 and25.

TABLE 3.3 Genetic distance of in vivo matured Nanobodies versus parentalV-germline sequences. Target interaction Number Number potency of AA ofAA in Nano- Nanobody MFI FACS mutations CDR3 body variant Llama (pre-(absorption versus different family within germ- Myc ratio; parental toDNA- (llama) family line Library incubation) ELISA) V-germline¹ clone  2(417) 1C9 VHH1e DNA 19 6 19 0 1A8 DNA 18 21 20 0 20C9 PB 97 11 20 020B10 PB 85 8 19 0  5 (418) 14F6 VHH2a/b DNA nd 15 16 0 8B4 DNA 71 13 170 8H5 DNA 24 12 18 0 6A11 DNA + PB 53 17 18 0 8E6 DNA 69 12 19 0 14G4DNA nd 34 19 0 8F5 DNA 143 16 15 2 8H6 DNA 43 17 15 2 8H4 DNA 65 27 17 214F10 PB nd 19 17 1 14G11 PB nd 30 18 2 8G11 PB 166 26 19 1 8G12 PB 16636 16 1 8D10 PB 94 18 16 1 8H10 PB 94 19 16 1 8B12 PB 93 38 16 1 8C12 PB89 28 15 1 6H10 PB 44 7 15 0 8A11 PB 17 11 16 1 15 (414) 16D9 VHH-3DNA + PB 53 7 10 0 19C2 DNA 64 18 10 0 19E3 DNA 55 8 10 0 1G6 DNA 11 1211 0 25 (407) 18C1 VHH-1e DNA 63 18 15 0 18A7 PB 69 14 14 2 ¹Any change,including a deletion or an addition are considered to calculate thenumber of mutations.

The average adsorption ratios (ELISA) of 20, 37 and 29 Nanobody variantsoriginating respectively from the ‘DNA’, ‘PB’ and ‘Cell’ repertoires,are 13, 13 and 26, respectively (FIG. 21), indicating superiority of‘Cell’ originating Nanobodies for this target. These observations arealso reflected at the monoclonal Nanobody level, as both most potentbinders (13A7, 7D5 and 7E2, example 3.10) the most potent blocker (13A7)were identified from the ‘Cell’ repertoire. However, when consideringindividual families, for family 5 (but not the others) a clear increaseis observed in average potency of the PB versus the DNA repertoire (23vs 18 adsorption ratios, and 95 vs. 66 mean fluorescence intensityFACS). This suggests that affinity maturation occurred as result of thecell boost in this family. In the other three families, the cell boostdid not improve potency. In the ranking of the potencies of thefunctional Nanobodies, however, the most potent enhancer 14D5 originatesfrom the DNA repertoire, while the second best blocker was derived fromfamily 5 from the PB repertoire. Both families were identified fromllama 418, which was the llama showing the strongest HcAb mediated serumresponse after DNA immunization (FIG. 13B).

Example 3.15. Genetic Immunizations with a Cocktail of Targets AllowIdentification of Nanobodies Specific for Each Individual Target

Immunization of outbred animals such as llama resulted in highlyvariable target specific humoral response magnitudes between theindividual animals. This effect is even more pronounced after geneticimmunization with low immunogenic cell surface expressed receptorscontaining multiple transmembrane domains such as GPCRs and ion channels(examples 2.4 and 3.1). It is expected that a detectable HcAb mediatedtarget specific response will at least increase the efficiency of thediscovery. Consequently, genetic immunization of outbred animals withtarget cocktails of any type (cell bound and non-cell bound molecules)would be favourable: a higher number of animals increases the chances toidentify more target responders (of high magnitude) without burdeningthe availability. As an example of cocktail DNA vaccination, weimmunized two llamas (405 and 418) with two ion channels P2X7 of mouseand human origin as described under example 3.2. Since the amino acididentity between mouse and human P2X7 is sufficiently different (80.5%sequence identity) and all mouse P2X7 identified Nanobodies lackcross-reactivity with human P2X7 (example 3.10), mouse and human P2X7can be utilised as relevant target examples to demonstrate the cocktailgenetic immunization approach.

In order to identify hP2X7 specific Nanobodies, cell based selectionswere performed to enrich for hP2X7 specific Nanobodies essentially asdescribed under example 3. Selections were performed on pooled ‘DNA’(PBL1+2) and ‘PB’ (PBL2+PBL3+LN) libraries from each llama 405 and 418to maintain all diversity, but keeping the libraries separate peranimal. Stable hP2X7 transfected YAC-1 cells andhP2X7-Art2.2-transfected CHO cells were used for the first and secondround of selection, respectively. After the second selection round, theenrichment was calculated as the ratio of the number of eluted phagefrom hP2X7 transfected cells versus non transfected CHO cells.Enrichment was 15- and 7-fold for libraries from llamas 405 and 418,respectively, indicating the presence of hP2X7 Nanobodies and suggestingsuccessful identification of Nanobodies against both ion channels.

After infection of E. coli TG1 with phage outputs after hP2X7 selection,individual clones will be picked and periplasmic extracts will beprepared as in example 1.4. The periplasmic extracts will be used todetermine specificity for human P2X7 via flow cytometry, similarly asperformed for mouse P2X7 (example 3.5). Periplasmic extracts showingclear staining on a hP2X7 transfected cell line (such as CHO or HEK293),while not above background on an identical parental cell linetransfected with mouse P2X7 or the same non-transfected WT cell line,will be considered to be specific for hP2X7. hP2X7 specificity will beconfirmed in a dose-dependent way using purified Nanobody via the samemethod to determine EC50s.

Example 4.1. Induction of a Humoral CXCR7 Specific Serum Titer FollowingGenetic Immunization of Llama Using the Helios Gene-Gun

As a second GPCR example to identify target specific Nanobodies aftergenetic immunization, the chemokine receptor CXCR7 was chosen. HumanCXCR7 encoding cDNA was cloned in pVAX1 and plasmid DNA purified asdescribed in example 2.1. After transfection, the pVAX1-CXCR7 constructshowed expression of native CXCR7 at the cell surface, confirmed bydifferential staining of CXCR7 transfected versus parental WT cellsfollowing a similar detection method to the one described in example 2.2(data not shown).

Four llamas (391, 395, 396 and 397) were immunized via intradermal jetinjection as described in example 2.4. A single 2×10⁷ cells CAKI/hCXCR7injection was administered 42 to 56 days after the fourth DNAimmunization. Three llamas (385, 387 and 404) were immunized with 4injections of 2×10⁷ stably transfected CXCR7/HEK293 cells with 14-dayintervals. The CXCR7 specific serum IgG titer was determined similarlyas in example 2.4 using pre-immune and immune serum samples after DNAvaccination and after the single cell boost. None of the seven llamasshowed a CXCR7 specific humoral response following the two immunizationregimes.

In another immunization experiment, four llamas (434, 439, 441 and 444)were genetically immunized using a Helios Gene-gun as described underexample 3. In short, 4 doses of DNA were applied (24 shots per dose) attwo-weekly intervals followed by a single cell boost of CAKI transfectedCXCR7 cells (2E7 cells). A pre-immune blood sample; one 8 days after thefourth DNA administration and a final blood sample 8 days after thesingle cell boost was collected from each llama and the target specificserum titer was determined via FACS as described under example 2.4. Outof four llamas immunized, only a single llama (444) showed a moderatethough consistently increased MCF with the ‘DNA’ and post-cell boostserum sample on CXCR7-transfected HEK293 cells compared to thepre-immune level for all dilutions tested (FIG. 23). In parallel, fourllamas (435, 436, 437, 440) were immunized with CXCR7-transfected CAKIcells by four cell injections (2×10⁷ cells per injection) at intervalsof 2 weeks. Contrary to llama 444, none of the latter four llamas showeda detectable CXCR7 serum titer.

Example 4.2. Identification of Target Specific Nanobodies in Absence ofa Detectable CXCR7 Specific Serum Conversion

Libraries were constructed from immune tissues collected after thegenetic immunization and after the cell boost (DNA and PB repertoire,respectively) for each of the llamas 391, 395, 396 and 397. Threeadditional libraries were constructed from the cell immunized llamas385, 387 and 404. An overview of the libraries is summarized in Table4.1. Phage display selections were performed with all 11 libraries on 10(selection round 1 and round 2) and 1 (selection round 2) units of CXCR7Membrane vesicles as described before (example 2.6). 853 clones from allround 2 selection outputs were screened (between 31 and 155 individualclones per library); results are summarized in Table 4.1. CXCR7specificity was determined via phage ELISA on 2 units of CXCR7 Membranevesicles applying 10-fold dilutions of phage supernatant prepared from a1 mL culture. 234 Nanobodies, showing a minimally 2-fold increased ELISAsignal on hCXCR7 Membrane vesicles over non-transfected control Membranevesicles, were considered to be CXCR7 specific. Round 2 hit-rates peranimal and repertoire are summarized in Table 4.1, ranging from 4 to61%. Based on the average hit-rates on round 2 selection outputsfollowing the different immunization strategies, discovery efficiencieswere calculated as 29, 28 and 26% for ‘DNA’, ‘PB’ and ‘cell’repertoires, respectively (FIG. 24). Even in absence of a detectableHcAb titer to CXCR7, target specific Nanobodies were identified from allimmunization strategies (DNA, PB, cell). All 234 CXCR7 specificNanobodies were sequenced and redundant Nanobodies (identical AAsequence) were removed. This resulted in the identification of 78 uniquesequences, belonging to 46 distinct Nanobody B-cell lineages (Table4.1). The number of variants (minimally 1 AA residue difference)identified within a family ranged between 1 and 12. The average numberof CXCR7 specific Nanobody families identified per llama is 5.3 aftercell immunizations (385, 387, 404) and 7.5 via DNA immunization(DNA+cell boost; 391, 395, 396, 397), respectively, showing that forthis target, genetic immunization (DNA+PB) resulted in a higher Nanobodyfamily diversity as compared to the diversity obtained via cellimmunizations.

TABLE 4.1 Discovery overview of CXCR7 specific Nanobody families. Numberof different CXCR7 specific Specificity Nanobody CXCR7 screeningfamilies (of Llama response Hit-rate which some ID Immunogen (FACS)Library (ELISA) are displacing) 391 DNA — DNA 26% (12/46) 4 CXCR7/CAKI —PB 18% (8/44) 2 395 DNA — DNA 45% (62/138) 6 CXCR7/CAKI — PB 29% (9/31)4 396 DNA — DNA 17% (8/46) 3 CXCR7/CAKI — PB 61% (22/36) 2 397 DNA — DNA17% (24/138) 6 CXCR7/CAKI — PB 10% (4/42) 3 385 CXCR7/Hek293 — Cells 46%(71/155) 7 387 CXCR7/Hek293 — Cells  4% (2/45) 2 404 CXCR7/Hek293 —Cells  9% (12/132) 7

To identify receptor function modulating Nanobodies, an SDF-1 liganddisplacement assay was performed using CXCR7/HEK293 membrane extractssimilarly as described for CXCR4 (example 2.7). Nanobodies showing aclear reduction in residual [¹²⁵I]-SDF-1 binding to CXCR7/HEK293membrane extracts were considered to be ligand competitors. The numberof Nanobody families scored as ligand displacer are illustrated in Table4.1, showing that ligand competing Nanobodies were identifiedirrespective of the immunization strategy (represented by repertoiresDNA, PB and cell).

Example 4.3. Cell Boost Following Genetic Immunization Generates aDifferent Nanobody Repertoire

From llamas 391, 395, 396 and 397, 29 CXCR7 specific Nanobody familieswere identified consisting of 55 Nanobody sequence variants (out of 234sequenced). Two families contained minimally 1 identical variant couldbe identified both in the DNA and the PB repertoire of llama 396, incontrasts to the findings in examples 2 (CXCR4) and 3 (P2X7). Only for 2of these 29 families (7% of the total family diversity), Nanobodyvariants belonging to the same family were identified from the ‘DNA’ and‘PB’ library (Table 4.1 and FIG. 25), again suggesting that the Nanobodyrepertoire after DNA vaccination (DNA) or cell boost (PB) is different.Genetic immunization only results in a different Nanobody repertoire,indicating that a part of the repertoire is not identified or lost afterthe cell boost.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

All of the references described herein are incorporated by reference, inparticular for the teaching that is referenced hereinabove.

The invention claimed is:
 1. An immunoglobulin that specifically bindsto P2X7, which consists of 4 framework regions (FR1 to FR4 respectively)and 3 complementarity determining regions (CDR1 to CDR3 respectively),in which: CDR1 is the amino acid sequence of SEQ ID NO: 279; CDR2 is theamino acid sequence of SEQ ID NO: 443; and CDR3 is the amino acidsequence of SEQ ID NO:
 607. 2. The immunoglobulin according to claim 1,wherein the immunoglobulin has at least 80% amino acid identity with theframework regions of the amino acid sequence of SEQ ID NO:
 778. 3. Animmunoglobulin that specifically binds to P2X7, that consists of SEQ IDNO:
 778. 4. A polypeptide comprising an immunoglobulin according toclaim
 1. 5. A polypeptide comprising at least two immunoglobulins,wherein at least one of the at least two immunoglobulins is theimmunoglobulin according to claim
 1. 6. The polypeptide according toclaim 5, wherein the polypeptide has at least 80% amino acid identitywith the framework regions of the amino acid sequence of SEQ ID NO: 790.7. A polypeptide that specifically binds to P2X7, that consists of SEQID NO:
 790. 8. An immunoglobulin that specifically binds to P2X7,wherein the immunoglobulin has at least 80% amino acid identity with theframework regions of the amino acid sequence of SEQ ID NO: 778, andwherein one or more of the amino acid residues at positions 11, 37, 44,45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering arechosen from the Hallmark residues listed in Table B-2.
 9. Thepolypeptide according to claim 5, wherein the polypeptide has at least90% amino acid identity with the framework regions of the amino acidsequence of SEQ ID NO:
 790. 10. The polypeptide according to claim 5,wherein the polypeptide has at least 95% amino acid identity with theframework regions of the amino acid sequence of SEQ ID NO:
 790. 11. Thepolypeptide according to claim 5, wherein the polypeptide has at least99% amino acid identity with the framework regions of the amino acidsequence of SEQ ID NO: 790.